Method for Producing an Acidic Substance Having a Carboxyl Group

ABSTRACT

An acidic substance having a carboxyl group is produced by culturing in a medium a microorganism which has been modified to enhance expression of the ybjL gene, and collecting the acidic substance having a carboxyl group from the medium.

This application is a Divisional of, and claims priority under 35 U.S.C.§120 to, U.S. patent application Ser. No. 12/579,577, filed Oct. 15,2009, which was a Continuation under 35 U.S.C. §120 of PCT PatentApplication No. PCT/JP2008/057478, filed Apr. 17, 2008, which claimedpriority under 35 U.S.C. §119 to Japanese Patent Application No.2007-108631, filed on Apr. 17, 2007, and Japanese Patent Application No.2007-242859, filed on Sep. 19, 2007, which are incorporated in theirentireties by reference herein. Also, the Sequence Listing filedelectronically herewith is hereby incorporated by reference (File name:2015-05-28T_US-408D_Seq_List; File size: 240 KB; Date recorded: May 28,2015).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for producing an acidicsubstance having a carboxyl group. L-Glutamic acid and L-aspartic acidare widely used as raw materials in making seasonings and so forth.Succinic acid is widely used as a raw material in making seasonings andbiodegradable plastics.

2. Brief Description of the Related Art

L-Glutamic acid is mainly produced by fermentation utilizing L-glutamicacid-producing bacteria of the so-called coryneform bacteria belongingto the genus Brevibacterium, Corynebacterium or Microbacterium, or theirmutant strains (see, for example, Kunihiko Akashi et al., Amino AcidFermentation, Japan Scientific Societies Press (Gakkai Shuppan Center),pp. 195-215, 1986). Methods are known for producing L-glutamic acid byfermentation using other bacterial strains, including microorganismsbelonging to the genera Bacillus, Streptomyces, Penicillium or the like(see, for example, U.S. Pat. No. 3,220,929), microorganisms belonging tothe genera Pseudomonas, Arthrobacter, Serratia, Candida or the like(see, for example, U.S. Pat. No. 3,563,857), microorganisms belonging tothe genera Bacillus, Pseudomonas, Serratia, Aerobacter aerogenes(currently referred to as Enterobacter aerogenes) or the like (see, forexample, Japanese Patent Publication (KOKOKU) No. 32-9393), a mutantstrain of Escherichia coli (see, for example, Japanese Patent Laid-open(KOKAI) No. 5-244970), and the like. In addition, methods for producingL-glutamic acid have also been disclosed using microorganisms belongingto the genera Klebsiella, Erwinia, Pantoea or Enterobacter (see, forexample, Japanese Patent Laid-open No. 2000-106869 (U.S. Pat. No.6,682,912), Japanese Patent Laid-open No. 2000-189169 (U.S. PatentPublished Application No. 2001009836), Japanese Patent Laid-open No.2000-189175 (U.S. Pat. No. 7,247,459)).

Furthermore, various techniques have been disclosed for increasing theL-glutamic acid-producing ability by enhancing the L-glutamic acidbiosynthetic enzymes using recombinant DNA techniques. For example, ithas been reported for Corynebacterium or Brevibacterium bacteria thatintroduction of a gene coding for citrate synthase derived fromEscherichia coli or Corynebacterium glutamicum was effective to enhancethe L-glutamic acid-producing ability of coryneform bacteria (see, forexample, Japanese Patent Publication No. 7-121228). Furthermore, it hasalso been reported that introduction of a citrate synthase gene derivedfrom a coryneform bacterium into enterobacteria belonging to the generaEnterobacter, Klebsiella, Serratia, Erwinia or Escherichia was effectiveto enhance the bacteria's L-glutamic acid-producing ability (see, forexample, Japanese Patent Laid-open No. 2000-189175 (U.S. Pat. No.7,247,459)).

Methods for improving the production of target substances such as aminoacids are also known, including by modifying the uptake or secretionsystems of target substances. Such methods include, for example, bydeleting or attenuating the system for uptake of a target substance intocells. Specifically, known methods to improve production of L-glutamicacid include deleting the gluABCD operon, or a part thereof, toeliminate or attenuate uptake of L-glutamic acid into cells (see, forexample, European Patent Application Laid-open No. 1038970), andenhancing production of purine nucleotides by attenuating uptake ofpurine nucleotides into cells (see, for example, European PatentApplication Laid-open No. 1004663), and the like.

Furthermore, methods of enhancing the secretion system for a targetsubstance, and methods of deleting or attenuating the secretion systemfor an intermediate or substrate in the biosynthetic system of a targetsubstance are known. Known methods of enhancing the secretion system ofa target substance include, for example, production of L-lysine byutilizing a Corynebacterium strain in which the L-lysine secretion gene(lysE) is enhanced (see, for example, WO2001/5959), and production ofL-glutamic acid by using an enterobacterium in which the L-glutamic acidsecretion system gene (yhfK) is enhanced (see, for example, JapanesePatent Laid-open No. 2005-278643 (U.S. Patent Published Application No.2005196846)). Furthermore, methods for producing an L-amino acid usingthe rhtA, B, C genes, which have been suggested to be involved in thesecretion of L-amino acids, have also been reported (see, for example,Japanese Patent Laid-open No. 2000-189177 (U.S. Patent PublishedApplication No. 2005239177)). Known methods for, for example, deleting asecretion system for an intermediate or substrate in a biosynthesissystem of a target substance include, for L-glutamic acid, mutating ordisrupting the 2-oxoglutarate permease gene to attenuate secretion of2-oxoglutarate, which is an intermediate of the target substance (see,for example, WO97/23597).

Furthermore, use of the gene coding for the ATP binding cassettesuperfamily (ABC transporter), which is involved in transportation ofsubstances through cell membranes, in the breeding of microorganisms inwhich transmembrane transportation of amino acids is modified has beensuggested (see, for example, WO00/37647).

Furthermore, it has also been reported that L-glutamic acid productionefficiency can be improved in Escherichia bacteria by enhancing theexpression of genes thought to participate in secretion of L-amino acidssuch as yfiK (see, for example, Japanese Patent Laid-open No.2000-189180 (U.S. Pat. No. 6,979,560)). Moreover, it has also beenreported that L-glutamic acid-producing ability can be improved byenhancing expression of the yhfK gene (see, for example, Japanese PatentLaid-open No. 2005-278643 (U.S. Patent Published Application No.2005196846)).

Moreover, methods are known for producing L-glutamic acid by culturing amicroorganism under acidic conditions to precipitate the L-glutamic acid(see, for example, Japanese Patent Laid-open No. 2001-333769 (U.S.Patent Published Application No. 2007134773)). When the pH is kept low,L-glutamic acid is precipitated, and the ratio of L-glutamic acid infree form with no electrical charge increases. As a result, theL-glutamic acid easily penetrates cell membranes. When L-glutamic acidis taken up into cells, it is converted into an intermediate of the TCAcycle, 2-oxoglutaric acid, in one step by glutamate dehydrogenase, andtherefore it is generally thought that L-glutamic acid taken up intocells is easily metabolized. However, 2-oxoglutarate dehydrogenaseactivity can be deleted or attenuated, or the like, in the fermentativeproduction of L-glutamic acid (for example, Japanese Patent Laid-openNo. 2001-333769 (U.S. Patent Published Application No. 2007134773),Japanese Patent Laid-open No. 7-203980 (U.S. Pat. No. 5,573,945)), butthen 2-oxoglutaric acid is not degraded, intracellular 2-oxoglutaricacid concentration increases which inhibits the growth of themicroorganism. Thus, the culture fails. Therefore, as described inJapanese Patent Laid-open No. 2001-333769 (U.S. Patent PublishedApplication No. 2007134773), a strain was bred using a mutation that isdeficient in 2-oxoglutarate dehydrogenase activity and can produceL-glutamic acid accompanying precipitation, and this strain can be usedfor the production of L-glutamic acid.

For fermentative production of non-amino organic acids, includingsuccinic acid, anaerobic bacteria including those belonging to the genusAnaerobiospirillum or Actinobacillus are usually used (U.S. Pat. Nos.5,142,834 and 5,504,004, Guettler, M. V. et al., 1999, InternationalJournal of Systematic Bacteriology, 49:207-216). Although the use ofsuch anaerobic bacteria provides high product yields, many nutrients arerequired for sufficient proliferation, and therefore, it is necessary toadd large amounts of organic nitrogen sources such as corn steep liquor(CSL) into the culture medium. The addition of large amounts of organicnitrogen sources can result in not only an increase in the cost of theculture, but also an increase in the cost for isolating or purifying theproduct, and therefore, their use is not economical.

In addition, methods are known in which aerobic bacteria such ascoryneform bacteria are cultured once under aerobic conditions, thenharvested, washed, and allowed to rest, producing a non-amino organicacid in the absence of supplied oxygen (Japanese Patent Laid-open Nos.11-113588 and 11-196888). These methods are economical since a smalleramount of organic nitrogen can be added, and the bacteria willsufficiently grow in a simple culture medium. However, there is still aroom for improvement in terms of production amount, concentration, andproduction rate per cell of the target organic acids, as well assimplification of the production process, and the like. Furthermore, theproduction of a non-amino organic acid by fermentation using a bacteriumin which phosphoenolpyruvate carboxylase activity is enhanced (forexample, Japanese Patent Laid-open No. 11-196887), and the like havealso been reported.

Furthermore, as for Escherichia coli, which is a facultative anaerobicgram negative bacterium, methods for producing a non-amino organic acidby culturing it once under aerobic conditions, and then allowing thecells to rest in the absence of supplied oxygen, resulting in ananaerobically produced non-amino organic acid (Vemuri G. N. et al.,2002, Journal of Industrial Microbiology and Biotechnology,28(6):325-332). This is similar to the methods using coryneformbacteria. Alternatively, E. coli can be aerobically cultured toaerobically produce the non-amino organic acid (U.S. Patent PublishedApplication No. 20050170482). However, since Escherichia coli is a gramnegative bacterium, it is vulnerable to osmotic pressure, and thereremains room for improvement in productivity per cell etc.

The ybjL gene is located on the genome of Escherichia coli (see, forexample, Blattner, F. R. et al., 1997, Science, 277(5331):1453-74), andit is also thought to code for a transporter on the basis of the motifs,topology etc. of the deduced amino acid sequence. However, cloning ofthe gene, as well as expression of the gene and analysis of theexpression product have not been reported, and the actual functions ofthe gene remained unknown.

SUMMARY OF THE INVENTION

An aspect of the present invention is to provide a bacterial strain thatcan efficiently produce an acidic substance having a carboxyl group,especially L-glutamic acid, L-aspartic acid, and succinic acid, and toprovide a method for efficiently producing an acidic substance having acarboxyl group by using such a strain.

The ybjL gene was isolated and shown to be involved in L-glutamic acidresistance. Also, it has been found that when the expression of the ybjLgene is enhanced, L-glutamic acid fermentation yield is improved and theproduction rate or yield of succinic acid is improved.

It is an aspect of the present invention to provide a microorganism thathas an ability to produce an acidic substance having a carboxyl groupand has been modified to enhance expression of the ybjL gene.

It is a further aspect of the present invention to provide theaforementioned microorganism, wherein enhanced expression is obtained bya method selected from the group consisting of: A) increasing copynumber of the ybjL gene, B) modifying an expression control sequence ofthe ybjL gene, and C) combinations thereof.

It is a further aspect of the present invention to provide theaforementioned microorganism, wherein the ybjL gene encodes a protein:(A) a protein comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, 4 and 87; (B) a protein comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 2, 4 and87, but wherein one or several amino acid residues are substituted,deleted, inserted or added, and the protein improves the ability of themicroorganism to produce an acidic substance having a carboxyl groupwhen expression of the gene encoding the protein is enhanced in themicroorganism.

It is a further aspect of the present invention to provide theaforementioned microorganism, wherein the ybjL gene encodes the proteinselected from the group consisting of SEQ ID NO: 5 and 88.

It is a further aspect of the present invention to provide theaforementioned microorganism, wherein the microorganism is a bacteriumbelonging to the family Enterobacteriaceae.

It is a further aspect of the present invention to provide theaforementioned microorganism, wherein the bacterium belongs to a genusselected from the group consisting of Escherichia, Enterobacter,Raoultella, Pantoea, and Klebsiella.

It is a further aspect of the present invention to provide theaforementioned microorganism, wherein the microorganism is a rumenbacterium.

It is a further aspect of the present invention to provide theaforementioned microorganism, wherein the microorganism is Mannheimiasucciniciproducens.

It is a further aspect of the present invention to provide theaforementioned microorganism, wherein the acidic substance is an organicacid selected from the group consisting of succinic acid, fumaric acid,malic acid, oxalacetic acid, citric acid, isocitric acid, α-ketoglutaricacid, and combinations thereof.

It is a further aspect of the present invention to provide theaforementioned microorganism, wherein the acidic substance is L-glutamicacid and/or L-aspartic acid.

It is a further aspect of the present invention to provide a method forproducing an acidic substance having a carboxyl groupcomprisingculturing the aforementioned microorganism in a medium to produce andaccumulate the acidic substance having a carboxyl group in the medium,and collecting the acidic substance having a carboxyl group from themedium.

It is a further aspect of the present invention to provide theaforementioned method, wherein the acidic substance is an organic acidselected from the group consisting of succinic acid, fumaric acid, malicacid, oxalacetic acid, citric acid, isocitric acid, α-ketoglutaric acid,and combinations thereof.

It is a further aspect of the present invention to provide theaforementioned method, wherein the acidic substance is L-glutamic acidand/or L-aspartic acid.

It is a further aspect of the present invention to provide a method forproducing an acidic substance having a carboxyl group comprising: A)allowing a substance to act on an organic raw material in a reactionmixture containing carbonate ions, bicarbonate ions, or carbon dioxidegas, wherein the substance is selected from the group consisting of i)the microorganism as described above, ii) a product obtained byprocessing the microorganism of i), and iii) combinations thereof, andcollecting the acidic substance having a carboxyl group.

It is a further aspect of the present invention to provide theaforementioned method, wherein the acidic substance is an organic acidselected from the group consisting of succinic acid, fumaric acid, malicacid, oxalacetic acid, citric acid, isocitric acid, α-ketoglutaric acid,and combinations thereof.

It is a further aspect of the present invention to provide a method forproducing a polymer comprising succinic acid comprising A) producingsuccinic acid by the aforementioned method, and B) polymerizing thesuccinic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of helper plasmid RSF-Red-TER.

FIG. 2 shows construction of helper plasmid RSF-Red-TER.

FIG. 3 shows the growth of the ybjL-amplified strain in the presence ofa high concentration L-glutamic acid. C.T. (hr) stands for ‘culturetime’ in hours. OD_(660 nm) stands for the optical density at 660 nm,which is a measure of growth of the respective strains of bacteria asidentified in the graph.

FIG. 4 shows accumulation of succinic acid obtained with theybjL-amplified strain of Escherichia bacterium (open squares) vs.control (closed circles).

FIG. 5 shows accumulation of succinic acid obtained with theybjL-amplified strain of Enterobacter bacterium (open squares) vs.control (closed circles).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the present invention will be explained in detail.

<1> Microorganism Having Ability to Produce Acidic Substance Having aCarboxyl Group

The microorganism in accordance with the presently disclosed subjectmatter has an ability to produce an acidic substance having a carboxylgroup and has been modified so that expression of the ybjL gene isenhanced. The term “ability to produce an acidic substance having acarboxyl group” can mean the ability of a microorganism to produce andcause accumulation of an acidic substance having a carboxyl group in amedium or cells to such a degree that the acidic substance having acarboxyl group can be collected from the cells or medium when themicroorganism of the present invention is cultured in the medium. Themicroorganism can originally have the ability to produce an acidicsubstance having a carboxyl group, or the ability to produce an acidicsubstance can be obtained by modifying a microorganism such as thosedescribed below using mutation or recombinant DNA techniques. Also, amicroorganism can be imparted with the ability to produce an acidicsubstance having a carboxyl group, or the ability to produce an acidicsubstance having a carboxyl group can be enhanced by introducing thegene described herein.

In the present invention, the “acidic substance having a carboxyl group”can mean an organic compound having one or more carboxyl groups andwhich is acidic when the substance is in the free form, and not in thesalt form. Specifically, examples include organic acids and L-aminoacids having two carboxyl groups, for example, acidic amino acids.Examples of the L-amino acids include L-glutamic acid and L-asparticacid, and examples of the organic acids include succinic acid, fumaricacid, malic acid, oxalacetic acid, citric acid, isocitric acid,α-ketoglutaric acid, and the like.

The parent strain which can be used to derive the microorganism inaccordance with the presently disclosed subject matter is notparticularly limited, and can include bacteria, for example,Enterobacteriaceae, rumen bacteria, and coryneform bacteria.

The Enterobacteriaceae family encompasses bacteria belonging to thegenera Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea,Photorhabdus, Providencia, Raoultella, Salmonella, Serratia, Shigella,Morganella, Yersinia, and the like. In particular, bacteria classifiedinto the family Enterobacteriaceae according to the taxonomy used by theNCBI (National Center for Biotechnology Information) database(www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) areexamples. Among these, bacteria belonging to the genus Escherichia,Enterobacter, Raoultella, Pantoea, Klebsiella, or Serratia areparticular examples.

A “bacterium belonging to the genus Escherichia” means that thebacterium is classified into the genus Escherichia according to theclassification known to a person skilled in the art of microbiology,although the bacterium is not particularly limited. Examples of thebacterium belonging to the genus Escherichia include, but are notlimited to, Escherichia coli (E. coli). Other examples include, forexample, the bacteria of the phyletic groups described in the work ofNeidhardt et al. (Neidhardt F. C. Ed., 1996, Escherichia coli andSalmonella: Cellular and Molecular Biology/Second Edition, pp.2477-2483, Table 1, American Society for Microbiology Press, Washington,D.C.). Specific examples include Escherichia coli W3110 (ATCC 27325),Escherichia coli MG1655 (ATCC 47076), and the like, and others derivedfrom the prototype wild-type strain, the K12 strain.

These strains are available from, for example, the American Type CultureCollection (Address: 12301 10801 University Boulevard, Manassas, Va.20108, United States of America). That is, registration numbers aregiven to each of the strains, and the strains can be ordered by usingthese numbers. The registration numbers of the strains are listed in thecatalogue of the American Type Culture Collection.

Pantoea, Erwinia, and Enterobacter bacteria are classified asγ-proteobacteria, and they are taxonomically very close to one another(J. Gen. Appl. Microbiol., 1997, 43, 355-361; Int. J. Syst. Bacteriol.,1997, 43, 1061-1067). In recent years, some bacteria belonging to thegenus Enterobacter were reclassified as Pantoea agglomerans, Pantoeadispersa, or the like, on the basis of DNA-DNA hybridization experimentsetc. (Int. J. Syst. Bacteriol., 1989, 39:337-345). Furthermore, somebacteria belonging to the genus Erwinia were reclassified as Pantoeaananas or Pantoea stewartii (refer to Int. J. Syst. Bacteriol., 1993,43:162-173).

Examples of the Enterobacter bacteria include Enterobacter agglomerans,Enterobacter aerogenes, and the like. Specifically, the strainsexemplified in European Patent Application Laid-open No. 952221 can beused. Typical strains of the genus Enterobacter include Enterobacteragglomerans ATCC 12287, Enterobacter aerogenes ATCC 13048, Enterobacteraerogenes NBRC 12010 (Biotechnol Bioeng., 2007, Mar. 27; 98(2):340-348),and Enterobacter aerogenes AJ110637 (FERM ABP-10955), and the like. TheAJ110637 strain was obtained from the soil at the seashore of SusukiKaigan, Makinohara-shi, Shizuoka-ken on March, 2006 by cumulative liquidculture using glycerol as the carbon source. The full-length 16S rDNAsequence was then determined, and it was found to be 99.9% homologous tothat from Enterobacter aerogenes NCTC 10006. Moreover, in aphysiological test using an API kit, the strain gave results similar tothe prototype species of Enterobacter aerogenes, and therefore it wasidentified as Enterobacter aerogenes. This strain was deposited atInternational Patent Organism Depository, Agency of Industrial Scienceand Technology (Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi,Ibaraki-ken, 305-8566, Japan) on Aug. 22, 2007, and assigned anaccession number of FERM P-21348. Then, the deposit was converted to aninternational deposit based on the Budapest Treaty on Mar. 13, 2008, andassigned an accession number of FERM ABP-10955.

Typical strains of the Pantoea bacteria include Pantoea ananatis,Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Specificexamples include the following strains:

Pantoea ananatis AJ13355 (FERM BP-6614, European Patent Laid-open No.0952221)

Pantoea ananatis AJ13356 (FERM BP-6615, European Patent Laid-open No.0952221)

Although these strains are described as Enterobacter agglomerans inEuropean Patent Laid-open No. 0952221, they are currently classified asPantoea ananatis on the basis of nucleotide sequence analysis of the 16SrRNA etc., as described above.

Examples of the Erwinia bacteria include Erwinia amylovora and Erwiniacarotovora, examples of the Klebsiella bacteria include Klebsiellaplanticola, and examples of the Raoultella bacteria include Raoultellaplanticola. Specific examples include the following strains:

Erwinia amylovora ATCC 15580 strain

Erwinia carotovora ATCC 15713 strain

Klebsiella planticola AJ13399 strain (FERM BP-6600, European PatentLaid-open No. 955368)

Klebsiella planticola AJ13410 strain (FERM BP-6617, European PatentLaid-open No. 955368).

Raouhella planticola ATCC 33531 strain

Although the AJ13399 strain and the AJ13410 strain were classified asKlebsiella planticola at the time of the deposit, Klebsiella planticolais currently classified into Raoultella planticola (Drancourt, M., 2001,Int. J. Syst. Evol. Microbiol, 51:925-32).

Examples of rumen bacteria include Mannheimia, Actinobacillus,Anaerobiospirillum, Pyrobacterium, and Selenomonas. Bacteria includingMannheimia succiniciproducens, Actinobacillus succinogenes, Selenomonasruminantium, Veillonella parvula, Wolnella succino genes,Anaerobiospirillum succiniciproducens, and the like, can be used.Specific strains include Mannheimia sp. 55E (KCTC0769BP strain, U.S.Patent Published Application No. 20030113885, International PatentPublication WO2005/052135).

<1-1> Imparting an Ability to Produce an Acidic Substance Having aCarboxyl Group

Hereinafter, methods for imparting an ability to produce an acidicsubstance having a carboxyl group to bacteria, or methods for enhancingthe ability to produce an acidic substance having a carboxyl group aredescribed.

To impart an ability to produce an acidic substance having a carboxylgroup, methods conventionally employed in the breeding of bacteria forproducing substances by fermentation (see “Amino Acid Fermentation”,Japan Scientific Societies Press, 1st Edition, published May 30, 1986,pp. 77-100) can be applied. Such methods include the acquisition of anauxotrophic mutant, an analogue-resistant strain, or a metabolicregulation mutant, or construction of a recombinant strain havingenhanced expression of an enzyme involved in biosynthesis of acidicsubstances having a carboxyl group. In the breeding of bacteria thatproduce an acidic substance having a carboxyl group, one or moreproperties, such as auxotrophic mutation, analogue resistance, ormetabolic regulation mutation, can be imparted. The expression of one ortwo or more enzymes involved in the biosynthesis of an acidic substancehaving a carboxyl group can be enhanced. Furthermore, the impartation ofproperties such as auxotrophic mutation, analogue resistance, ormetabolic regulation mutation can be combined with the enhancement ofthe biosynthetic enzymes.

A mutant strain auxotrophic for an acidic substance having a carboxylgroup, a strain resistant to an analogue of an acidic substance having acarboxyl group, or a metabolic regulation mutant strain can be obtainedby subjecting a parent or wild-type strain to a conventionalmutagenesis, such as exposure to X-rays or UV irradiation, or atreatment with a mutagen such as N-methyl-N′-nitro-N-nitrosoguanidine,and then selecting bacteria which exhibit an autotrophy, analogueresistance, or metabolic regulation mutation and which also have theability to produce an acidic substance having a carboxyl group.

Methods for imparting an amino acid- or organic acid-producing abilityto microorganisms, and amino acid- or organic acid-producing bacteriawill be specifically exemplified below.

<L-Glutamic Acid-Producing Bacteria>

Examples of the method of modifying a microorganism to impart L-glutamicacid-producing ability to the microorganism include, for example,enhancing expression of a gene coding for an enzyme involved inL-glutamic acid biosynthesis. Examples of an enzyme involved inL-glutamic acid biosynthesis include glutamate dehydrogenase(“GDH”)(gdh), glutamine synthetase (glnA), glutamate synthetase (gltAB),isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB),citrate synthase (“CS”)(gltA), methylcitrate synthase (hereinafter alsoreferred to as “PRPC” (prpC), phosphoenolpyruvate carboxylase (“PEPC”)(ppc), pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, lpdA),pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA),enolase (eno), phosphoglyceromutase (pgmA, pgml), phosphoglyceratekinase (pgk), glyceraldehyde-3-phophate dehydrogenase (gapA), triosephosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp),phosphofructokinase (pfkA, pfkB), and glucose phosphate isomerase (pgi),and the like. The capital letter abbreviations in parentheses after theenzyme names are the enzyme abbrevation, while the lower caseabbreviations indicate the name of the gene encoding the enzyme.

Methods for modifying a microorganism to increase expression of a targetgene will be explained below.

The first method is to increase the copy number of the target gene bycloning the target gene on an appropriate plasmid and transforming thechosen host bacterium with the obtained plasmid. For example, when thetarget gene is the gene coding for CS (gltA gene), the gene coding forPEPC (ppc gene) or the gene coding for GDH (gdhA gene), nucleotidesequences of these genes from Escherichia bacteria and Corynebacteriumbacteria have already been reported (Ner, S. et al., 1983, Biochemistry,22:5243-5249; Fujita, N. et al., 1984, J. Biochem., 95:909-916; Valle,F. et al., 1984, Gene, 27:193-199; Microbiology, 140:1817-1828, 1994;Eikmanns, B. J. et al., 1989, Mol. Gen. Genet., 218:330-339; Bormann, E.R. et al., 1992, Molecular Microbiology, 6:317-326), and therefore theycan be obtained by synthesizing primers based on their respectivenucleotide sequences, and performing PCR using chromosomal DNA as thetemplate.

Examples of plasmids which can be used for transformation include aplasmid which autonomously replicates in the host bacterium belonging tothe family Enterobacteriaceae, such as pUC19, pUC18, pBR322, RSF1010,pHSG299, pHSG298, pHSG399, pHSG398, pSTV28, pSTV29 (pHSG and pSTV areavailable from Takara Bio), pMW119, pMW118, pMW219, pMW218 (pMW vectorsare available from Nippon Gene), and the like. Moreover, a phage DNA canalso be used as the vector instead of a plasmid. Examples of plasmidsfor simultaneously enhancing the activities of CS or PRPC, PEPC, and GDHas described above include RSFCPG which includes the gltA gene, ppcgene, and gdhA gene (refer to European Patent Laid-open No. 0952221),and RSFPPG which is the same as RSFCPG, but the gltA gene is replacedwith the prpC gene.

Examples of transformation methods include treating recipient cells withcalcium chloride so to increase permeability for DNA, which has beenreported for Escherichia coli K-12 (Mandel, M. and Higa, A., 1970, J.Mol. Biol., 53:159-162), and preparing competent cells from cells whichare in the growth phase, followed by transformation with DNA, which hasbeen reported for Bacillus subtilis (Duncan, C. H., et al., 1977, Gene,1:153-167). Alternatively, a method of making potential host cells intoprotoplasts or spheroplasts, which can easily take up recombinant DNA,followed by introducing the recombinant DNA into the cells. This methodis known to be applicable to Bacillus subtilis, actinomycetes and yeasts(Chang, S. and Choen, S. N., 1979, Molec. Gen. Genet., 168:111-115;Bibb, M. J. et al., 1978, Nature, 274:398-400; Hinnen, A., et al., 1978,Proc. Natl. Sci., USA, 75:1929-1933). In addition, microorganisms canalso be transformed by the electric pulse method (Japanese PatentLaid-open No. 2-207791).

The copy number of a target gene can also be increased by introducingmultiple copies of the gene into the chromosomal DNA of themicroorganism. Multiple copies of a gene can be introduced into thechromosomal DNA by homologous recombination (Experiments in MolecularGenetics, 1972, Cold Spring Harbor Laboratory) using multiple copies ofa sequence as targets in the chromosomal DNA. Sequences, which arepresent in multiple copies on the chromosomal DNA, repetitive DNAs, andinverted repeats present at the end of a transposable element, areexemplary. Also, as disclosed in Japanese Patent Laid-open No. 2-109985,it is possible to incorporate a target gene into a transposon, and allowit to be transferred to introduce multiple copies of the gene into thechromosomal DNA. The target gene can also be introduced into thebacterial chromosome by using Mu phage (Japanese Patent Laid-open No.2-109985).

The second method is to increase expression of the target gene byreplacing an expression regulatory sequence of the target gene, such aspromoter, on the chromosomal DNA or plasmid with a stronger promoter.For example, the lac promoter, trp promoter, trc promoter, etc. areknown as strong promoters. Moreover, it is also possible to substituteseveral nucleotides in the promoter region of a gene so that thepromoter is stronger, or to modify the SD sequence as disclosed inInternational Patent Publication WO00/18935. Examples of methods forevaluating the strength of promoters and strong promoters are describedin the article of Goldstein et al. (Goldstein, M. A., and Doi, R. H.,1995, Biotechnol. Annu. Rev., 1:105-128), etc.

Substitution of an expression regulatory sequence can be performed, forexample, in the same manner as for gene substitution using atemperature-sensitive plasmid. Examples of vectors having atemperature-sensitive replication origin and are usable for Escherichiacoli and Pantoea ananatis include, for example, the pMAN997 plasmiddescribed in International Publication WO99/03988, and the like.Furthermore, an expression regulatory sequence can also be substitutedby the method called “Red-driven integration” using Red recombinase of λphage (Datsenko, K. A. and Wanner, B. L., 2000, Proc. Natl. Acad. Sci.USA. 97:6640-6645), or by combining the Red-driven integration methodand the λ phage excisive system (Cho, E. H., et al., 2002, J.Bacteriol., 184:5200-5203) (WO2005/010175), and the like. Modificationof an expression regulatory sequence can be combined with increasing thegene copy number.

As shown in Reference Example 1, a strain resistant to a λ Red geneproduct, for example, Pantoea ananatis SC17(0), can be used for the Reddriven integration. The SC17(0) strain was deposited at the RussianNational Collection of Industrial Microorganisms (VKPM), GNII Genetika(Russia, 117545 Moscow 1, Dorozhny proezd. 1) on Sep. 21, 2005 under anaccession number of VKPM B-9246.

Examples of microorganisms modified by the method described above sothat expression of citrate synthase gene, methylcitrate synthase gene,phosphoenolpyruvate carboxylase gene and/or glutamate dehydrogenase geneare enhanced include the microorganisms disclosed in Japanese PatentLaid-open Nos. 2001-333769, 2000-106869, 2000-189169, 2000-333769,2006-129840, WO2006/051660, and the like.

Furthermore, L-glutamic acid-producing ability can also be imparted byenhancing the 6-phosphogluconate dehydratase activity,2-keto-3-deoxy-6-phosphogluconate aldolase activity, or both. Examplesof the microorganism in which 6-phosphogluconate dehydratase activityand 2-keto-3-deoxy-6-phosphogluconate aldolase activity are increasedinclude the microorganism disclosed in Japanese Patent Laid-open No.2003-274988.

L-glutamic acid-producing ability can also be imparted by decreasing oreliminating the activity of an enzyme that catalyzes a reaction thatbranches off from the L-glutamic acid biosynthesis pathway, producing acompound other than L-glutamic acid. Examples of these include2-oxoglutarate dehydrogenase (sucA), isocitrate lyase (aceA), phosphateacetyltransferase (pta), acetate kinase (ack), acetohydroxy acidsynthase (ilvG), acetolactate synthase (ilvI), formate acetyltransferase(pfl), lactate dehydrogenase (ldh), glutamate decarboxylase (gadAB),1-pyrroline-5-carboxylate dehydrogenase (putA), and the like. Gene namesare indicated in the parentheses after the enzyme names. The activity of2-oxoglutarate dehydrogenase can be decreased or completely eliminated.

In order to decrease or eliminate the activities of the aforementionedenzymes, methods similar to the method for decreasing or eliminating theactivity of lactate dehydrogenase (LDH) described later can be used.

A decrease in the intracellular activity of the target enzyme and thedegree by which the activity is decreased, including if it is completelyeliminated, can be confirmed by measuring the enzyme activity of a cellextract or a purified fraction thereof obtained from a candidate strainand comparing it with that of a wild-type strain. For example,2-oxoglutarate dehydrogenase activity can be measured by the method ofReed et al. (Reed L. J. and Mukherjee B. B., 1969, Methods inEnzymology, 13, pp. 55-61).

Methods of eliminating or decreasing the 2-oxoglutarate dehydrogenaseactivity in Escherichia bacteria are disclosed in Japanese PatentLaid-open Nos. 5-244970, 7-203980 and the like. A method of eliminatingor decreasing 2-oxoglutarate dehydrogenase activity in coryneformbacteria is disclosed in International Patent Publication WO95/34672.Furthermore, such a method for Enterobacter bacteria is disclosed inJapanese Patent Laid-open No. 2001-333769.

Specific examples of Escherichia bacteria which are deficient in2-oxoglutarate dehydrogenase activity or in which the 2-oxoglutaratedehydrogenase activity is decreased include the following strains (U.S.Pat. Nos. 5,378,616 and 5,573,945).

E. coli W3110sucA::Kmr

E. coli AJ12624 (FERM BP-3853)

E. coli AJ12628 (FERM BP-3854)

E. coli AJ12949 (FERM BP-4881)

E. coli W3110sucA::Kmr is obtained by disrupting the 2-oxoglutaratedehydrogenase gene (sucA gene) of Escherichia coli W3110. This strain iscompletely deficient in 2-oxoglutarate dehydrogenase.

Other specific examples of bacteria wherein the activity of2-oxoglutarate dehydrogenase is deleted or decreased include thefollowing strains:

Pantoea ananatis AJ13601 (FERM BP-7207, European Patent Laid-open No.1078989)

Pantoea ananatis AJ13356 (FERM BP-6615, U.S. Pat. No. 6,331,419)

Pantoea ananatis SC17sucA (FERM BP-8646, WO2005/085419)

Klebsiella planticola AJ13410 strain (FERM BP-6617, U.S. Pat. No.6,197,559)

Brevibacterium lactofermentum ΔS strain (refer to International PatentPublication WO95/34672)

The SC17sucA strain is obtained by obtaining a low phlegm productionmutant strain (SC17) from the AJ13355 strain, which was isolated fromnature as a strain that could proliferate in a medium containingL-glutamic acid and a carbon source at low pH, and disrupting the2-oxoglutarate dehydrogenase gene (sucA) in the mutant strain. TheAJ13601 strain is obtained by introducing the plasmid RSFCPG containingthe gltA, ppc and gdhA genes derived from Escherichia coli and theplasmid pSTVCB containing the gltA gene derived from Brevibacteriumlactofermentum into the SC17sucA strain to obtain theSC17sucA/RSFCPG+pSTVCB strain, and selecting a high concentrationL-glutamic acid-resistant strain at a low pH and a strain showing a highproliferation degree and a high L-glutamic acid-producing ability(European Patent Laid-open No. 0952221). The AJ13356 strain was obtainedby deleting the αKGDH-E1 subunit gene (sucA) from the AJ13355 strain.

The SC17sucA strain was assigned a private number of AJ417, deposited inthe International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology (Central 6, 1-1, Higashi1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, postal code: 305-8566) on Feb.26, 2004, and assigned an accession number of FERM BP-08646.

The AJ13410 strain was deposited in the International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology (Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken,Japan, postal code: 305-8566) on Feb. 19, 1998, and assigned anaccession number of FERM P-16647. The deposit was then converted to aninternational deposition under the provisions of Budapest Treaty on Jan.11, 1999 and assigned an accession number of FERM BP-6617.

The aforementioned Pantoea ananatis AJ13355, AJ13356, and AJ13601strains and the Klebsiella planticola AJ13399 strain have an ability toaccumulate L-glutamic acid in a concentration which exceeds thesaturation concentration of L-glutamic acid in a liquid medium when itis cultured under acid conditions.

Furthermore, in order to improve the L-glutamic acid-producing abilityof Enterobacteriaceae bacteria, the method of deleting the arcA gene(U.S. Pat. No. 7,090,998), and the method of amplifying the yhfK gene,which is a glutamic acid secretion gene (WO2005/085419) can also beused.

Other than the above, examples of coryneform bacteria having L-glutamicacid-producing ability include the following strains:

Brevibacterium flavum AJ11573 (FERM P-5492, refer to Japanese PatentLaid-open No. 56-151495)

Brevibacterium flavum AJ12210 (FERM P-8123, refer to Japanese PatentLaid-open No. 61-202694)

Brevibacterium flavum AJ12212 (FERM P-8123, refer to Japanese PatentLaid-open No. 61-202694)

Brevibacterium flavum AJ12418 (FERM-BP2205, refer to Japanese PatentLaid-open No. 2-186994)

Brevibacterium flavum DH18 (FERM P-11116, refer to Japanese PatentLaid-open No. 3-232497)

Corynebacterium melassecola DH344 (FERM P-11117, refer to JapanesePatent Laid-open No. 3-232497)

Corynebacterium glutamicum AJ11574 (FERM P-5493, refer to JapanesePatent Laid-open No. No. 56-151495)

Microorganisms having L-glutamic acid-producing ability include theBrevibacterium lactofermentum AJ13029 strain (FERM BP-5189, refer toWO96/06180), which was made to be able to produce L-glutamic acid in amedium containing an excessive amount of biotin in the absence of biotinaction inhibitor, such as surfactant, by introducing a mutation whichimparts temperature sensitivity to the biotin action inhibitor. Examplesfurther include microorganisms that belong to the genus Alicyclobacillusand are resistant to a L-glutamic acid antimetabolite (Japanese PatentLaid-open No. 11-262398).

Furthermore, the microorganism having a L-glutamic acid producingability can also be unable to degrade L-glutamic acid, or express themaleate synthase (aceB).isocitrate lyase (aceA).isocitrate dehydrogenasekinase/phosphatase (aceK) operon (henceforth abbreviated as “aceoperon”) constitutively. Examples of such microorganisms include, forexample, the following:

Escherichia coli AJ12628 (FERM BP-3854)

Escherichia coli AJ12624 (FERM BP-3853)

The former is a mutant strain in which 2-oxoglutarate dehydrogenaseactivity is decreased and expression of the ace operon has becomeconstitutive. The latter is a mutant strain in which 2-oxoglutaratedehydrogenase activity is decreased and the ability to degradeL-glutamic acid is decreased (refer to French Patent No. 2680178).

When a Pantoea bacterium is used, a mutation can be imparted thatresults in production of less extracellular phlegm as compared to awild-type strain when it is cultured in a medium containing asaccharide. In order to introduce such a mutation, bacterial strains canbe screened for their ability to produce viscous materials on the solidmedium (Japanese Patent Laid-open No. 2001-333769), and the ams operonthat is involved in polysaccharide synthesis can be disrupted. Thenucleotide sequence of the ams operon is shown in SEQ ID NO: 66, and theamino acid sequences of AmsH, I, A, C and B encoded by this operon areshown in SEQ ID NOS: 67, 68, 69, 70 and 71, respectively.

<Succinic Acid-Producing Bacteria>

As succinic acid-producing bacteria, strains which are unable to formacetic acid, lactic acid, ethanol, and formic acid can be used, andspecific examples include the Escherichia coli SS373 strain(International Patent Publication WO99/06532).

Strains deficient in their abilities to form acetic acid, lactic acid,ethanol and formic acid can be obtained by using a strain that cannotassimilate acetic acid and lactic acid in a minimal medium, or bydecreasing the activities of the lactic acid or acetic acid biosyntheticenzymes described below (International Patent PublicationWO2005/052135).

Moreover, such strains as described above can also be obtained byimparting monofluoroacetic acid resistance (U.S. Pat. No. 5,521,075).

Other examples of methods for obtaining a strain with improved succinicacid-producing ability include culturing a strain which is deficient inboth formate lyase and lactate dehydrogenase and cannot assimilatepyruvic acid in a glucose-enriched medium under anaerobic conditions,and isolating a mutant strain having the ability to assimilate pyruvicacid (International Patent Publication WO97/16528).

The ability to produce succinic acid can also be imparted by amplifyinga gene encoding an enzyme which is involved in the succinic acidbiosynthesis system described below, or deleting a gene encoding anenzyme which catalyzes a reaction which branches away from the succinicacid biosynthesis system to produce another compound.

The ability to produce succinic acid can also be imparted by modifying amicroorganism to decrease the enzymatic activity of lactatedehydrogenase (LDH), which is a lactic acid biosynthesis system enzyme(International Patent Publications WO2005/052135, WO2005/116227, U.S.Pat. No. 5,770,435, U.S. Patent Published Application No. 20070054387,International Patent Publication WO99/53035, Alam, K. Y. and Clark, D.P., 1989, J. Bacteriol., 171:6213-6217). Some microorganisms can haveboth L-lactate dehydrogenase and D-lactate dehydrogenase, and can bemodified to decrease the activity of either one of the enzymes, or theactivities of both the enzymes, in another example.

The ability to produce succinic acid can also be imparted by modifying amicroorganism to decrease the enzymatic activity of the formic acidbiosynthesis system enzyme, pyruvate-formate lyase (PFL) (U.S. PatentPublished Application No. 20070054387, International Patent PublicationsWO2005/116227, WO2005/52135, Donnelly, M. I. et al., 1998, Appl.Biochem. Biotechnol., 70-72:187-198).

The ability to produce succinic acid can also be imparted by modifying amicroorganism to decrease the enzymatic activities of phosphateacetyltransferase (PTA), acetate kinase (ACK), pyruvate oxidase (POXB),acetyl-CoA synthetase (ACS) and acetyl-CoA hydrolase (ACH), which areacetic acid biosynthesis system enzymes (U.S. Patent PublishedApplication No. 20070054387, International Patent PublicationsWO2005/052135, WO99/53035, WO2006/031424, WO2005/113745, andWO2005/113744).

The ability to produce succinic acid can also be enhanced by modifying amicroorganism to decrease the enzymatic activity of alcoholdehydrogenase (ADH), which is an ethanol biosynthesis system enzyme(refer to International Patent Publication WO2006/031424).

The ability to produce succinic acid can also be enhanced by decreasingthe activities of pyruvate kinase, glucose PTS (ptsG), ArcA protein,IclR protein (iclR), glutamate dehydrogenase (gdh) and/or glutaminesynthetase (glnA), and glutamate synthase (gltBD) (International PatentPublication WO2006/107127, WO2007007933, Japanese Patent Laid-open No.2005-168401). The gene names are indicated in the parentheses followingthe enzyme names.

The ability to produce succinic acid can also be imparted by enhancing abiosynthesis system enzyme involved in the succinic acid production.

The ability to produce succinic acid can also be enhanced by increasingthe enzymatic activities of pyruvate carboxylase, malic enzyme,phosphoenolpyruvate carboxylase, fumarase, fumarate reductase, malatedehydrogenase and phosphoenolpyruvate carboxykinase (Japanese PatentLaid-open No. 11-196888, International Patent Publication WO99/53035,Hong, S. H., and S. Y. Lee, 2001, Biotechnol. Bioeng., 74:89-95,Millard, C. S. et al., 1996, Appl. Environ. Microbiol., 62:1808-1810,International Patent Publication WO2005/021770, Japanese PatentLaid-open No. 2006-320208, Kim, P. et al., 2004, Appl. Environ.Microbiol., 70:1238-1241). Increasing the enzymatic activities of thesetarget enzymes can be performed by referring to the methods forenhancing expression of a target gene described herein, and the methodsfor enhancing expression of ybjL gene described later.

Specific examples of succinic acid-producing bacteria belonging to thefamily Enterobacteriaceae include the following strains:

Escherichia coli SS373 strain (International Patent PublicationWO99/06532)

Escherichia coli AFP111 strain (International Patent PublicationWO97/16528)

Escherichia coli NZN111 strain (U.S. Pat. No. 6,159,738)

Escherichia coli AFP184 strain (International Patent PublicationWO2005/116227)

Escherichia coli SBS100MG strain, SBS110MG strain, SBS440MG strain,SBS550MG strain, and SBS660MG strain (International Patent PublicationWO2006/031424)

Examples of succinic acid-producing bacteria belonging to coryneformbacteria include the following strains.

Brevibacterium flavum AB-41 strain (Japanese Patent Laid-open No.11-113588)

Brevibacterium flavum AB-41 strain (PC-amplified strain, Japanese PatentLaid-open No. 11-196888)

Corynebacterium glutamicum AJ110655 strain (FERM BP-10951)

Brevibacterium flavum MJ233A1dh strain (International Patent PublicationWO2005/021770)

Brevibacterium lactofermentum (Corynebacterium glutamicum) 2256Δ(ldh,ach, pta, ack) (International Patent Publication WO2005/113744)

Brevibacterium lactofermentum 2256Δ(ldh, pta, ack, poxB) (InternationalPatent Publication WO2005/113745)

Corynebacterium glutamicum Rldh-/pCRB-1 PC strain (International PatentPublication WO2005/010182)

Examples of succinic acid-producing rumen bacteria include the followingstrains.

Mannheimia succiniciproducens LPK, LPK7 and LPK4 (International PatentPublication WO2005/052135)

Actinobacillus succinogenes 130Z (U.S. Pat. No. 5,504,004)

Anaerobiospirillum succiniciproducens FZ10 (U.S. Pat. No. 5,521,075)

Anaerobiospirillum succiniciproducens FZ53 (U.S. Pat. No. 5,573,931)

<1-2> Enhancement of Expression of ybjL Gene

A microorganism that has an ability to produce an acidic substancehaving a carboxyl group can be modified such as those described above sothat expression of the ybjL gene is enhanced. However, the modificationto enhance expression of the ybjL gene is performed first, and then theability to produce an acidic substance having a carboxyl group can beimparted. Furthermore, the microorganism can already have the ability toproduce an acidic substance having a carboxyl group, or the ability toproduce an acidic substance having a carboxyl group can be enhanced byamplification of the ybjL gene.

The “ybjL gene” refers to the ybjL gene of Escherichia coli or ahomologue thereof, the ybjL gene of Pantoea ananatis or a homologuethereof, or the ybjL gene of Enterobacter aerogenes or a homologuethereof. Examples of the ybjL gene of Escherichia coli include a genecoding for a protein having the amino acid sequence of SEQ ID NO: 4, forexample, a gene having the nucleotide sequence of the nucleotides 101 to1783 in SEQ ID NO: 3. Furthermore, examples of the ybjL gene derivedfrom Pantoea ananatis include a gene coding for a protein having theamino acid sequence of SEQ ID NO: 2, for example, a gene having thenucleotide sequence of the nucleotides 298 to 1986 in SEQ ID NO: 1.Furthermore, examples of the ybjL gene derived from the Enterobacteraerogenes AJ110673 strain include a gene coding for a protein having theamino acid sequence of SEQ ID NO: 87, for example, a gene having thenucleotide sequence of the nucleotides 19 to 1704 in SEQ ID NO: 86.Furthermore, examples of the ybjL gene derived from Salmonellatyphimurium include a gene coding for a protein having the amino acidsequence of SEQ ID NO: 25 (SEQ ID NO: 24). Examples of the ybjL genederived from Yersinia pestis include a gene coding for a protein havingthe amino acid sequence of SEQ ID NO: 27 (SEQ ID NO: 26). Examples ofthe ybjL gene derived from Erwinia carotovora include a gene coding fora protein having the amino acid sequence of SEQ ID NO: 29 (SEQ ID NO:28). Examples of the ybjL gene derived from Vibrio cholerae include agene coding for a protein having the amino acid sequence of SEQ ID NO:31 (SEQ ID NO: 30). Examples of the ybjL gene derived from Aeromonashydrophila include a gene coding for a protein having the amino acidsequence of SEQ ID NO: 33 (SEQ ID NO: 32). Examples of the ybjL genederived from Photobacterium profundum include a gene coding for aprotein having the amino acid sequence of SEQ ID NO: 35 (SEQ ID NO: 34).Furthermore, the ybjL gene can be cloned from a coryneform bacteriumsuch as Corynebacterium glutamicum and Brevibacterium lactofermentum,Pseudomonas bacterium such as Pseudomonas aeruginosa, Mycobacteriumbacterium such as Mycobacterium tuberculosis or the like, on the basisof homology to the genes exemplified above. The amino acid sequences ofthe proteins encoded by the ybjL genes of Salmonella typhimurium,Yersinia pestis, Erwinia carotovora, Vibrio cholerae, Aeromonashydrophila and Photobacterium profundum described above are 96%, 90%,88%, 64%, 60% and 68% homologous to the amino acid sequence of SEQ IDNO: 4, respectively, and 86%, 90%, 84%, 63%, 60% and 67% homologous tothe amino acid sequence of SEQ ID NO: 2, respectively. The amino acidsequences of SEQ ID NOS: 2 and 4 are 86% homologous. The consensussequence of SEQ ID NOS: 2 and 4 is shown in SEQ ID NO: 5. The amino acidsequences of SEQ ID NOS: 4 and 87 re 92% homologous, and the amino acidsequences of SEQ ID NOS: 2 and 87 are 83% homologous. The sequence ofthe ybjL gene from the Enterobacter aerogenes AJ110637 strain (SEQ IDNO: 86) and the encoded amino acid sequence (SEQ ID NO: 87) have notbeen previously reported. The consensus sequence for the amino acidsequences of SEQ ID NOS: 2, 4 and 87 is shown in SEQ ID NO: 88.

The term “ybjL gene homologue” refers to a gene derived from amicroorganism other than Escherichia coli, Pantoea ananatis, andEnterobacter aerogenes, which exhibits high structural similarity to theybjL gene of Escherichia coli, Pantoea ananatis, or Enterobacteraerogenes and codes for a protein that improves an ability of amicroorganism to produce an acidic substance having a carboxyl groupwhen expression of the gene is enhanced in the microorganism. Examplesof ybjL gene homologues include genes coding for a protein having ahomology of 70% or more, 80% or more in another example, 90% or more inanother example, 95% or more in another example, 97% or more in anotherexample, to the entire amino acid sequence of SEQ ID NO: 2, 4 or 87 orthe amino acid sequence of SEQ ID NO: 2, 4 or 87, and which improves anability to produce an acidic substance having a carboxyl group of amicroorganism when expression of the gene is enhanced in themicroorganism. The ybjL gene homologue can code for a protein having ahomology of 70% or more, 80% or more in another example, 90% or more inanother example, 95% or more in another example, or 97% or more inanother example, to any of the aforementioned amino acid sequences, andthe consensus sequences of the SEQ ID NOS: 5 or 88. Homology of theamino acid sequences and nucleotide sequences can be determined byusing, for example, the algorithm BLAST of Karlin and Altschul (Pro.Natl. Acad. Sci. USA, 90, 5873 (1993)) or FASTA (Methods Enzymol., 183,63 (1990)). Programs called BLASTN and BLASTX were developed on thebasis of the algorithm BLAST (refer to www.ncbi.nlm.nih.gov). The term“homology” can also be used to mean “identity”.

The ybjL gene can have one or more conservative mutations, and can beartificially modified, so long as enhancement of the tbjL gene'sexpression results in an improved ability to produce a target substanceby a microorganism. That is, the ybjL gene can encode for an amino acidsequence of a known protein, for example, a protein having an amino acidsequence of SEQ ID NO: 2, 4, 25, 27, 29, 31, 33, 35 or 87, but whichincludes a conservative mutation, specifically, substitution, deletion,insertion or addition, of one or several amino acid residues at one orseveral positions. Although the number meant by the term “several” candiffer depending on the position in the three-dimensional structure ofthe protein, or the type of amino acid residue. For example, it can be 1to 20, 1 to 10 in another example, 1 to 5 in another example.Furthermore, the ybjL gene can encode for a protein having a homology of70% or more, 80% or more in another example, 90% or more in anotherexample, 95% or more in another example, or 97% or more in anotherexample, to the entire amino acid sequence of SEQ ID NO: 2, 4, 25, 27,29, 31, 33, 35 or 87, and which improves an ability to produce a targetsubstance by a microorganism when expression of the gene is enhanced inthe microorganism.

The aforementioned conservative substitution can be neutral, in that thefunction of the protein is not changed. The conservative mutation cantake place mutually among Phe, Trp and Tyr, if the substitution site isan aromatic amino acid; among Leu, Ile and Val, if the substitution siteis a hydrophobic amino acid; between Gln and Asn, if it is a polar aminoacid; among Lys, Arg and His, if it is a basic amino acid; between Aspand Glu, if it is an acidic amino acid; and between Ser and Thr, if itis an amino acid having hydroxyl group. Specific examples ofsubstitution considered conservative substitution include: substitutionof Ser or Thr for Ala; substitution of Gln, His or Lys for Arg;substitution of Glu, Gln, Lys, His or Asp for Asn; substitution of Asn,Glu or Gln for Asp; substitution of Ser or Ala for Cys; substitution ofAsn, Glu, Lys, His, Asp or Arg for Gln; substitution of Gly, Asn, Gln,Lys or Asp for Glu; substitution of Pro for Gly; substitution of Asn,Lys, Gln, Arg or Tyr for His; substitution of Leu, Met, Val or Phe forIle; substitution of Be, Met, Val or Phe for Leu; substitution of Asn,Glu, Gln, His or Arg for Lys; substitution of Ile, Leu, Val or Phe forMet; substitution of Trp, Tyr, Met, Ile or Leu for Phe; substitution ofThr or Ala for Ser; substitution of Ser or Ala for Thr; substitution ofPhe or Tyr for Trp; substitution of His, Phe or Trp for Tyr; andsubstitution of Met, Ile or Leu for Val.

Such a gene can be obtained by modifying the nucleotide sequence of thenucleotides 298 to 1986 in SEQ ID NO: 1, the nucleotide sequence of thenucleotides 101 to 1783 in SEQ ID NO: 3, the nucleotide sequence of thenucleotides 19 to 1704 in SEQ ID NO: 86, or the nucleotide sequence ofSEQ ID NO: 24, 26, 28, 30, 32 or 34 by, for example, site-specificmutagenesis, so that substitution, deletion, insertion or addition of anamino acid residue or residues occurs at a specific site in the encodedprotein. Furthermore, such a gene can also be obtained by a knownmutation treatment, examples of which include treating a gene having anyof the nucleotide sequences described above with hydroxylamine or thelike in vitro, and treating a microorganism, for example, an Escherichiabacterium, containing the gene with ultraviolet ray irradiation or amutagen used in a usual mutation treatment such asN-methyl-N′-nitro-N-nitrosoguanidine (NTG) or EMS (ethylmethanesulfonate). The mutation for the substitutions, deletions,insertions, additions, inversions or the like of amino acid residuesdescribed above also includes a naturally occurring mutation based onindividual difference, difference in species of microorganisms thatcontain the ybjL gene (mutant or variant), and the like.

The ybjL gene can also be a DNA hybridizable with a complementary strandof DNA having the nucleotide sequence of the nucleotides 298 to 1986 inSEQ ID NO: 1, a DNA having the nucleotide sequence of the nucleotides101 to 1783 in SEQ ID NO: 3, a DNA having the nucleotide sequence of thenucleotides 19 to 1704 in SEQ ID NO: 86, or a DNA having the nucleotidesequence of SEQ ID NO: 1, 3, 24, 26, 28, 30, 32, 34 or 86, or a probethat can be prepared from the DNAs having these sequences understringent conditions and which codes for a protein which improves anability to produce a substance of a microorganism when expression of thegene is enhanced in the microorganism.

The “stringent conditions” can be conditions under which a so-calledspecific hybrid is formed, and non-specific hybrid is not formed. It isdifficult to clearly express this condition by using any numericalvalue. However, the stringent conditions include, for example, when DNAshaving high homology to each other, for example, DNAs having a homologyof, for example, not less than 80%, not less than 90% in anotherexample, not less than 95% in another example, or not less than 97% inanother example, hybridize with each other, and DNAs having homologylower than the above level do not hybridize with each other, and whenwashing in ordinary Southern hybridization, i.e., washing once, twice orthree times in another example, at salt concentrations and temperatureof 1×SSC, 0.1% SDS at 60° C., 0.1×SSC, 0.1% SDS at 60° C. in anotherexample, 0.1×SSC, 0.1% SDS at 65° C., 0.1×SSC in another example, or0.1% SDS at 68° C. in another example.

The probe can have a partial sequence of the ybjL gene. Such a probe canbe produced by PCR using oligonucleotides prepared on the basis of thenucleotide sequence of the gene and a DNA fragment including the gene asthe template and performed in a manner well known to those skilled inthe art. When a DNA fragment having a length of about 300 bp is used asthe probe, the washing conditions after hybridization can be exemplifiedby 2×SSC, 0.1% SDS at 50° C.

The aforementioned descriptions concerning the gene homologue andconservative mutations can be similarly applied to the other enzymegenes described herein.

The expression of the ybjL gene can be enhanced by increasing the copynumber of the ybjL gene, modifying an expression control sequence of theybjL gene, amplifying a regulator that increases expression of the ybjLgene, or deleting or attenuating a regulator that decreases expressionof the ybjL gene. Expression can be enhanced or increased usingtransformation or homologous recombination performed in the same manneras the methods for enhancing expression of a target gene described abovefor L-glutamic acid-producing bacteria.

In the microorganism, an activity for secreting an acidic substancehaving a carboxyl group can be improved by enhancing the expression ofthe ybjL gene. Whether “the activity for secreting an acidic substancehaving a carboxyl group is improved” can be confirmed by comparing theamount of the acidic substance having a carboxyl group which has beensecreted into the medium in which the microorganism is cultured with theamount obtained by culturing a control microorganism into which the ybjLgene is not introduced. That is, the “improvement in the activity forsecreting the acidic substance having a carboxyl group” is observed asan increase in the concentration of the acidic substance having acarboxyl group in the medium in which the microorganism is cultured ascompared to the concentration obtained with a control microorganism.Furthermore, the “improvement in the activity for secreting an acidicsubstance having a carboxyl group” is also observed as a decrease inintracellular concentration of the acidic substance having a carboxylgroup in the microorganism. As for the improvement of the “activity forsecreting an acidic substance having a carboxyl group”, theintracellular concentration of the acidic substance having a carboxylgroup can be decreased by 10% or more, 20% or more in another example,30% or more in another example, as compared to the concentration in astrain in which expression of the ybjL gene is not enhanced. Theabsolute “activity for secreting an acidic substance having a carboxylgroup” of a microorganism can be detected by measuring the differencebetween the intracellular and extracellular concentrations of the acidicsubstance having a carboxyl group. Furthermore, the “activity forsecreting an acidic substance having a carboxyl group” can also bedetected by measuring the activity for taking up amino acids into thecells using reverted membrane vesicles with a radioisotope (J. Biol.Chem., 2002, vol. 277, No. 51, pp. 49841-49849). For example, theactivity can be measured by preparing reverted membrane vesicles fromcells in which the ybjL gene is expressed, adding a substrate which canact as a driving force such as ATP, and measuring the uptake activityfor RI-labeled glutamic acid. Moreover, the activity can also bemeasured by detecting an exchange reaction rate of a labeled acidicsubstance having a carboxyl group and unlabeled acidic substance havinga carboxyl group in live bacteria.

The microorganism can have an ability to accumulate L-glutamic acid inthe liquid medium in an amount which is more than the amount at thesaturation concentration of L-glutamic acid when it is cultured underacidic conditions (this ability is also referred to as the “L-glutamicacid accumulation ability under acidic conditions”). Such amicroorganism can have the L-glutamic acid accumulation ability underacidic conditions by enhancing the expression of the ybjL gene.Alternatively, the microorganism can have a native ability to accumulateL-glutamic acid under acidic conditions.

Specific examples of microorganisms having L-glutamic acid accumulationability under acidic conditions include the aforementioned Pantoeaananatis AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615),AJ13601 strain (FERM BP-7207) (for these, refer to Japanese PatentLaid-open No. 2001-333769), and the like. The Pantoea ananatis AJ13355and AJ13356 strains were deposited at the National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry (currently,National Institute of Bioscience and Human-Technology, NationalInstitute of Advanced Industrial Science and Technology, Address:Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566,Japan) on Feb. 19, 1998 and given accession numbers of FERM P-16644 andFERM P-16645. The deposits were then converted to international depositsunder the provisions of Budapest Treaty on Jan. 11, 1999 and givenaccession numbers of FERM BP-6614 and FERM BP-6615. The AJ13601 strainwas deposited at the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology(currently, International Patent Organism Depository, National Instituteof Advanced Industrial Science and Technology) on Aug. 18, 1999 andgiven an accession number of FERM P-17516. The deposit was converted toan international deposit under the provisions of the Budapest Treaty onJul. 6, 2000 and given an accession number of FERM BP-7207. Thesestrains were identified as Enterobacter agglomerans when they wereisolated and deposited as Enterobacter agglomerans AJ13355, AJ13356, andAJ13601 strains. However, they were recently re-classified as Pantoeaananatis on the basis of nucleotide sequencing of 16S rRNA and the like.

When an organic acid, especially succinic acid, is produced, using amicroorganism which is modified to decrease the activities of one ormore enzymes among lactate dehydrogenase (LDH), alcohol dehydrogenase(ADH), and pyruvate formate lyase (PFL), in addition to increasing theexpression of the ybjL gene, is more effective.

The expression “modified so that lactate dehydrogenase activity isdecreased” can mean that the lactate dehydrogenase activity is decreasedas compared to that of a strain in which lactate dehydrogenase isunmodified. The lactate dehydrogenase activity can be decreased to 10%per cell or lower as compared to that of a lactatedehydrogenase-unmodified strain. The lactate dehydrogenase activity canalso be completely deleted. Decrease of the lactate dehydrogenaseactivity can be confirmed by measuring the lactate dehydrogenaseactivity by a known method (Kanarek, L. and Hill, R. L., 1964, J. Biol.Chem., 239:4202). Specific examples of the method for producing a mutantstrain of Escherichia coli in which the lactate dehydrogenase activityis decreased include the method described in Alam, K. Y., and Clark, D.P., 1989, J. Bacteriol., 171:6213-6217, and the like. The microorganismwith decreased lactate dehydrogenase activity and enhanced expression ofthe ybjL gene can be obtained by, for example, preparing a microorganismin which the LDH gene is disrupted, and transforming this microorganismwith a recombinant vector containing the ybjL gene, as described inExample 1. However, either the modification for decreasing the LDHactivity or the modification for enhancing expression of the ybjL genecan be performed first. In Escherichia coli, LDH is encoded by the ldhAand lldD genes. The DNA sequence of the ldhA gene is shown in SEQ ID NO:36, the amino acid sequence encoded thereby is shown in SEQ ID NO: 37,the DNA sequence of the lldD gene is shown in SEQ ID NO: 38, and theamino acid sequence encoded thereby is shown in SEQ ID NO: 39.

In order to decrease or delete activity of LDH, a mutation thatdecreases or deletes the intracellular activity of LDH can be introducedinto the LDH gene on the chromosome by a known mutagenesis method. Forexample, the gene coding for LDH on the chromosome can be deleted, or anexpression control sequence such as a promoter and the Shine-Dalgarno(SD) sequence can be modified by gene recombination. Furthermore, amutation can also be introduced to cause an amino acid substitution(missense mutation), a stop codon (nonsense mutation), or a frame shiftmutation that adds or deletes one or two nucleotides into the codingregion of the LDH on the chromosome, or a part of, or the entire genecan be deleted (Qiu Z. and Goodman M. F., 1997, J. Biol. Chem.,272:8611-8617). Furthermore, the LDH activity can also be decreased ordeleted by gene disruption, for example, by deleting the coding regionof the LDH gene in a DNA construct, and replacing the normal LDH genewith the DNA construct on the chromosome by homologous recombination orthe like, or by introducing a transposon or IS factor into the gene.

In order to introduce a mutation which results in decreasing or deletingthe LDH activity by genetic recombination, for example, the followingmethods are used. The chromosomal LDH gene can be replaced with a mutantgene by preparing a mutant LDH gene in which a partial sequence of theLDH gene is modified so that it does not produce an enzyme that canfunction normally, and transforming a bacterium with a DNA containingthe mutant gene to cause homologous recombination between the mutantgene and the gene on a chromosome. Such site-specific mutagenesis basedon gene substitution utilizing homologous recombination has been alreadyreported, and include a method called Red driven integration developedby Datsenko and Wanner (Datsenko, K. A, and Wanner, B. L., 2000, Proc.Natl. Acad. Sci. USA, 97:6640-6645), a method of using a linear DNA suchas by utilizing Red driven integration in combination with an excisionsystem derived from λ phage (Cho, E. H., et al., 2002, J. Bacteriol.,184:5200-5203), a method of using a plasmid containing a temperaturesensitive replication origin (U.S. Pat. No. 6,303,383, Japanese PatentLaid-open No. 05-007491, WO2005/010175), and the like. Suchsite-specific mutagenesis based on gene substitution using homologousrecombination as described above can also be performed by using aplasmid that is unable to replicate in a host.

The expression “modified so that alcohol dehydrogenase activity isdecreased” can mean that the alcohol dehydrogenase activity is decreasedas compared to that of a strain in which alcohol dehydrogenase isunmodified. The alcohol dehydrogenase activity can be decreased to 10%per cell or lower as compared to an alcohol dehydrogenase-unmodifiedstrain. The alcohol dehydrogenase activity can also be completelydeleted. Decrease in the alcohol dehydrogenase activity can be confirmedby measuring the alcohol dehydrogenase activity by a known method(Lutstorf, U. M. et al., 1970, Eur. J. Biochem., 17:497-508). Specificexamples of the method for producing a mutant strain of Escherichia coliin which the alcohol dehydrogenase activity is decreased include themethod described in Sanchez A. M. et al., 2005, Biotechnol. Prog.,21:358-365, and the like. The microorganism in which alcoholdehydrogenase activity is decreased and expression of the ybjL gene isenhanced can be obtained by, for example, preparing a microorganism inwhich the gene coding for alcohol dehydrogenase (ADH) is disrupted, andtransforming this microorganism with a recombinant vector containing theybjL gene. However, either the modification for decreasing the ADHactivity or the modification for enhancing expression of the ybjL genecan be performed first. The alcohol dehydrogenase activity can bedecreased by a method similar to that for decreasing the lactatedehydrogenase activity described above. The nucleotide sequence (partialsequence) of the ADH gene from the Enterobacter aerogenes AJ110637strain (FERM ABP-10955) is shown in SEQ ID NO: 74. The entire nucleotidesequence of this gene can be determined by, for example, isolating theADH gene (adhE) from the chromosomal DNA of Enterobacter aerogenes onthe basis of this partial sequence.

The expression “modified so that pyruvate formate lyase activity isdecreased” can mean that the pyruvate formate lyase activity isdecreased as compared to that of a strain in which the pyruvate formatelyase is unmodified. The pyruvate formate lyase activity can bedecreased to 10% per cell or lower as compared to a pyruvate formatelyase-unmodified strain. The pyruvate formate lyase activity can also becompletely deleted. A decrease in the pyruvate formate lyase activitycan be confirmed by measuring the pyruvate formate lyase activity by aknown method (Knappe, J. and Blaschkowski, H. P., 1975, Meth. Enzymol.,41:508-518). The microorganism in which pyruvate formate lyase activityis decreased and expression of the ybjL gene is enhanced can be obtainedby, for example, preparing a microorganism in which the PFL gene isdisrupted, and transforming this microorganism with a recombinant vectorcontaining the ybjL gene. However, either the modification fordecreasing the PFL activity or the modification for enhancing expressionof ybjL can be performed first. The pyruvate formate lyase activity canbe decreased by a method similar to the method for decreasing thelactate dehydrogenase activity described above.

A bacterium modified so that the pyruvate carboxylase (PC) activity isenhanced, in addition to the enhanced the expression of the ybjL gene,can also be used to produce an organic acid, especially succinic acid.Enhancing the pyruvate carboxylase activity can be combined withdecreasing the lactate dehydrogenase activity, alcohol dehydrogenaseactivity, and/or pyruvate formate lyase activity. The expression“modified so that pyruvate carboxylase activity is enhanced” can meanthat the pyruvate carboxylase activity is increased as compared to thatof an unmodified strain such as a wild-type strain or parent strain. Thepyruvate carboxylase activity can be measured by, for example, a methodof measuring a decrease of NADH as described later.

The nucleotide sequence of the PC gene can be a reported sequence or canbe obtained by isolating a DNA fragment encoding a protein having the PCactivity from the chromosome of a microorganism, animal, plant, or thelike, and then the nucleotide sequence can be determined. After thenucleotide sequence is determined, a gene synthesized on the basis ofthat sequence can also be used.

The PC gene can be derived from a coryneform bacterium, such asCorynebacterium glutamicum (Peters-Wendisch, P. G. et al., 1998,Microbiology, vol. 144:915-927). Furthermore, so long as the functionsof the encoded PC protein, such as its involvement in carbon dioxidefixation, are not substantially degraded, nucleotides in the PC gene canbe replaced with other nucleotides or deleted, or other nucleotides canbe inserted into the sequence. Alternatively, a part of the nucleotidesequence can be transfered.

PC genes obtained from bacteria other than Corynebacterium glutamicum,including from other microorganisms, animals, and plants, can also beused. In particular, the sequences of PC genes derived from othermicroorganisms, animals and plants described below are known (referencesare indicated below), and they can be obtained by hybridization oramplification by PCR of the ORF portions in the same manner as describedabove.

Human [Biochem. Biophys. Res. Comm., 202, 1009-1014, (1994)]

Mouse [Proc. Natl. Acad. Sci. USA., 90, 1766-1779, (1993)]

Rat [GENE, 165, 331-332, (1995)]

Yeast: Saccharomyces cerevisiae [Mol. Gen. Genet., 229, 307-315,(1991)], Schizosaccharomyces pombe [DDBJ Accession No.; D78170]

Bacillus stearothermophilus [GENE, 191, 47-50, (1997)]

Rhizobium etli [J. Bacteriol., 178, 5960-5970, (1996)]

The PC gene can be enhanced in the same manner as when enhancingexpression of a target gene described above for L-glutamicacid-producing bacteria and enhancing expression of the ybjL gene.

<2> Method for Producing an Acidic Substance Having a Carboxyl Group

An acidic substance having a carboxyl group can be produced by culturingthe microorganism in a medium to produce and cause accumulation of anacidic substance having a carboxyl group in the medium and collectingthe substance from the medium. Furthermore, the acidic substance havinga carboxyl group can also be produced by allowing the microorganism, ora product obtained by processing the microorganism, to act on an organicraw material in a reaction mixture containing carbonate ions,bicarbonate ions, or carbon dioxide gas to produce the acidic substancehaving a carboxyl group, and collecting the substance. The former methodis exemplary for producing an acidic amino acid. The latter method isexemplary for producing an organic acid. Hereinafter, further examplesmethods for producing an acidic amino acid and an organic acid will beexemplified.

<2-1> Production of Acidic Amino Acid

An acidic amino acid can be produced by culturing the microorganism in amedium to produce and cause accumulation of an acidic amino acid in themedium and collecting the acidic amino acid from the medium.

As the medium used for the culture, a known medium containing a carbonsource, nitrogen source, and inorganic salts, as well as trace amountsof organic nutrients such as amino acids and vitamins as required can beused. Either a synthetic medium or natural medium can be used. Thecarbon source and nitrogen source used in the medium can be of any typeso long as substances that can be utilized by the chosen strain to becultured.

As the carbon source, saccharides such as glucose, glycerol, fructose,sucrose, maltose, mannose, galactose, starch hydrolysate and molassescan be used. In addition, alcohols such as ethanol can be usedindependently or in combination with another carbon source. Furthermore,organic acids such as acetic acid and citric acid other than the targetsubstance can also be used as the carbon source. As the nitrogen source,ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate,ammonium chloride, ammonium phosphate and ammonium acetate, nitrates andthe like can be used. As the trace amount organic nutrients, aminoacids, vitamins, fatty acids, nucleic acids, those containing thesesubstances such as peptone, casamino acid, yeast extract and soybeanprotein decomposition products can be used. When an auxotrophic mutantstrain that requires an amino acid or the like for growth is used, therequired nutrient can be supplemented. As mineral salts, phosphates,magnesium salts, calcium salts, iron salts, manganese salts and the likecan be used.

The culture can be performed as an aeration culture, while thefermentation temperature can be controlled to be 20 to 45° C., and pH tobe 3 to 9. When pH drops during the culture, the medium can beneutralized by addition of, for example, calcium carbonate, or with analkali such as ammonia gas. An acidic amino acid can accumulate in theculture broth, for example, after 10 to 120 hours of culture under suchconditions as described above.

When the acidic amino acid is L-glutamic acid, L-glutamic acid canprecipitate into the medium by using a liquid medium, the pH of which isadjusted so that L-glutamic acid precipitates. L-glutamic acidprecipitates around, for example, pH 5.0 to 4.0, pH 4.5 to 4.0 inanother example, pH 4.3 to 4.0 in another example, or pH 4.0 in anotherexample.

After completion of the culture, L-glutamic acid can be collected fromthe culture medium by any known collection method. For example, aftercells are removed from the culture medium, L-glutamic acid can becollected by concentrating the culture medium so it crystallizes, or byion exchange chromatography, or the like. When the culture is performedso that L-glutamic acid precipitates, the L-glutamic acid that hasprecipitated in the medium can be collected by centrifugation,filtration, or the like. In this case, it is also possible tocrystallize L-glutamic acid that has dissolved in the medium, and thencollect the precipitated L-glutamic acid in the culture broth togetherwith the crystallized L-glutamic acid.

<2-2> Production of Organic Acid

An organic acid can be produced by allowing the microorganism, or aproduct obtained by processing the microorganism, to act on an organicraw material in a reaction mixture containing carbonate ions,bicarbonate ions, or carbon dioxide gas, and collecting the organicacid.

In a first example, by culturing the microorganism in a mediumcontaining carbonate ions, bicarbonate ions, or carbon dioxide gas, andan organic raw material, proliferation of the microorganism andproduction of the organic acid simultaneously occur. In this example,the medium can be the reaction mixture. Proliferation of themicroorganism and production of the organic acid can simultaneouslyoccur, or there can be a period during the culture in whichproliferation of the microorganism mainly occurs, and a period in whichproduction of the organic acid mainly occurs.

In a second example, by allowing cells that have proliferated in themedium to coexist with a reaction mixture containing carbonate ions,bicarbonate ions, or carbon dioxide gas, and an organic raw material,and thereby allowing the microorganism to act on the organic rawmaterial in the reaction mixture, an organic acid can be produced. Inthis example, a product obtained by processing the cells of themicroorganism can also be used. Examples of the product obtained byprocessing cells include, for example, immobilized cells obtained withacrylamide, carragheenan, or the like, a disrupted cellular product, acentrifugation supernatant of the disrupted product, a fraction obtainedby partial purification of the supernatant by ammonium sulfate treatmentor the like, and the like.

Although the bacteria can be obtained on a solid medium such as an agarmedium by slant culture, bacteria previously cultured in a liquid medium(seed culture) are examples.

A medium usually used for culture of microorganisms can be used. Forexample, a typical medium obtained by adding natural nutrients such asmeat extract, yeast extract and peptone, to a composition comprisinginorganic salts such as ammonium sulfate, potassium phosphate andmagnesium sulfate can be used.

In the aforementioned first example, the carbon source added to themedium also serves as the organic raw material for the production of theorganic acid.

In the aforementioned second example, the cells after the culture arecollected by centrifugation, membrane separation, or the like, and usedfor the organic acid production reaction.

The organic raw material is not particularly limited so long as thechosen microorganism can assimilate it to produce succinic acid.However, fermentable carbohydrates including carbohydrates such asgalactose, lactose, glucose, fructose, glycerol, sucrose, saccharose,starch and cellulose, polyalcohols such as glycerin, mannitol, xylitoland ribitol, and the like are typically used. Glucose, fructose andglycerol are examples, and glucose is a particular example. When theorganic acid is succinic acid, fumaric acid or the like can be added inorder to efficiently produce succinic acid as described in JapanesePatent Laid-open No. 5-68576, and malic acid can be added instead offumaric acid.

Furthermore, a saccharified starch solution, molasses, or the likecontaining the aforementioned fermentable carbohydrates can also beused. The fermentable carbohydrates can be used independently or incombination. Although concentration of the aforementioned organic rawmaterial is not particularly limited, it is more advantageous that theconcentration is as high as possible and within such a range that theculture of the microorganism and production of the organic acid are notinhibited. In the aforementioned first example, the concentration of theorganic raw material in the medium is generally in the range of 5 to 30%(w/v), or 10 to 20% (w/v) in another example. Furthermore, in theaforementioned second example, the concentration of the organic rawmaterial in the reaction mixture is generally in the range of 5 to 30%(w/v), or 10 to 20% (w/v) in another example. Furthermore, it may benecessary to add additional organic raw material as the concentration ofthe organic raw material decreases with the progress of the culture orreaction.

The aforementioned reaction mixture containing carbonate ions,bicarbonate ions, or carbon dioxide gas and the organic raw material isnot particularly limited, and it can be, for example, a typical mediumfor culturing microorganisms, or it can be a buffer such as phosphatebuffer. The reaction mixture can be an aqueous solution containing anitrogen source, inorganic salts, and the like. The nitrogen source isnot particularly limited so long the chosen microorganism can assimilateit to produce an organic acid, and specific examples include variousorganic or inorganic nitrogen compounds such as ammonium salts,nitrates, urea, soybean hydrolysate, casein degradation products,peptone, yeast extract, meat extract, and corn steep liquor. Examples ofthe inorganic salts include various phosphates, sulfates, and metallicsalts such as those of magnesium, potassium, manganese, iron, and zinc.If necessary, growth-promoting factors including vitamins such asbiotin, pantothenic acid, inositol, and nicotinic acid, nucleotides,amino acids and the like can be added. In order to suppress foamingduring the reaction, an appropriate amount of a commercially availableantifoam can be added to the medium.

The pH of the reaction mixture can be adjusted by adding sodiumcarbonate, sodium bicarbonate, potassium carbonate, potassiumbicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide,magnesium hydroxide, or the like. Since the pH for the reaction isusually 5 to 10, or 6 to 9.5 in another example, the pH of the reactionmixture can be adjusted to be within the aforementioned range with analkaline substance, carbonate, urea, or the like, even during thereaction, if needed.

Water, buffer, medium or the like can be used as the reaction mixture,but a medium is a particular example. The medium can contain, forexample, the aforementioned organic raw material, and carbonate ions,bicarbonate ions, or carbon dioxide gas, and the reaction can beperformed under anaerobic conditions. Magnesium carbonate, sodiumcarbonate, sodium bicarbonate, potassium carbonate, or potassiumbicarbonate can be the source of the carbonate or bicarbonate ions, andthese can also be used as a neutralizing agent. However, if necessary,carbonate or bicarbonate ions can also be supplied from carbonic acid orbicarbonic acid or salts thereof or carbon dioxide gas. Specificexamples of salts of carbonic acid or bicarbonic acid include, forexample, magnesium carbonate, ammonium carbonate, sodium carbonate,potassium carbonate, ammonium bicarbonate, sodium bicarbonate, potassiumbicarbonate, and the like. Carbonate ions or bicarbonate ions can beadded at a concentration of 0.001 to 5 M, 0.1 to 3 M in another example,or 1 to 2 M in another example. When carbon dioxide gas is present, itcan be in an amount of 50 mg to 25 g, 100 mg to 15 g in another example,or 150 mg to 10 g in another example, per liter of the solution.

The optimal growth temperature of the chosen microorganism is generallyin the range of 25 to 40° C. Therefore, the reaction temperature isgenerally in the range of 25 to 40° C., or in the range of 30 to 37° C.in another example. The amount of bacterial cells in the reactionmixture is, although it is not particularly limited, 1 to 700 g/L, 10 to500 g/L in another example, or 20 to 400 g/L in another example. Thereaction time can be 1 to 168 hours, or 3 to 72 hours in anotherexample. The reaction can be performed batchwise or on a column.

The culture of the bacteria can be performed under aerobic conditions.On the other hand, the organic acid production reaction can be performedunder aerobic conditions, microaerobic conditions, or anaerobicconditions. When performed under microaerobic conditions or anaerobicconditions, the reaction can be performed in a sealed reaction vesselwithout aeration, with a supplied inert gas such as nitrogen gas, orwith a supplied inert gas containing carbon dioxide gas, and the like.

The organic acid can accumulate in the reaction mixture (culture medium)and can be separated and purified from the reaction mixture in aconventional manner. Specifically, solids such as bacterial cells can beremoved by centrifugation, filtration, or the like, then the resultingsolution can be desalted with an ion exchange resin or the like, and theorganic acid can be separated and purified from the solution bycrystallization or column chromatography.

Furthermore, when the target organic acid is succinic acid, and afterthe production, a polymerization reaction can be carried out by usingthe produced succinic acid as a raw material to produce a polymercontaining succinic acid. In recent years, with the increase ofenvironmentally friendly industrial products, polymers prepared from rawmaterials of plant origin have been attracting attention. The producedsuccinic acid can be converted into polymers such as polyesters andpolyamides and used (Japanese Patent Laid-open No. 4-189822). Specificexamples of succinic acid-containing polymers include succinic acidpolyesters obtained by polymerizing a diol such as butanediol andethylene glycol and succinic acid, succinic acid polyamides obtained bypolymerizing a diamine such as hexamethylenediamine and succinic acid,and the like. In addition, succinic acid and succinic acid-containingpolymers obtained by the production method described herein, andcompositions containing these can be used for food additives,pharmaceutical agents, cosmetics, and the like.

EXAMPLES

Hereinafter, the present invention will be explained more specificallywith reference to the following non-limiting examples.

Reference Example 1 Construction of Pantoea ananatis Strain Resistant toλ Red Gene Product

In order to disrupt the sdhA gene in Pantoea ananatis, a recipientstrain for efficiently carrying out the method called “Red-drivenintegration” or “Red-mediated integration” (Datsenko, K A. and Wanner,B. L., 2000, Proc. Natl. Acad. Sci. USA., 97, 6640-6645) wasconstructed.

First, the novel helper plasmid RSF-Red-TER which expresses the gam, betand exo genes of λ(“λ Red genes”) was constructed (FIG. 1). The detailsof this construction are described in Reference Example 2.

This plasmid can be used in a wide range of hosts having differentgenetic backgrounds. This is because 1) this plasmid has the replicon ofthe RSF1010 wide host spectrum plasmid (Scholz, et al., 1989, Gene,75:271-288; Buchanan-Wollaston et al., 1987, Nature, 328:172-175), whichis stably maintained by many types of gram negative and gram positivebacteria, and even plant cells, 2) the λ Red genes, gam, bet and exo,are under the control of the PlacUV5 promoter, which is recognized bythe RNA polymerases of many types of bacteria (for example, Brunschwig,E. and Darzins, A., 1992, Gene, 111, 1, 35-41; Dehio, M. et al, 1998,Gene, 215, 2, 223-229), and 3) the autoregulation factor PlacUV5-lacIand the ρ-non-dependent transcription terminator (TrrnB) of the rrnBoperon of Escherichia coli lower the basal expression level of the λ Redgenes (Skorokhodova, A. Y. et al, 2004, Biotekhnologiya (Rus), 5, 3-21).Furthermore, the RSF-Red-TER plasmid contains the levansucrase gene(sacB), and by the expression of this gene, the plasmid can be collectedfrom cells in a medium containing sucrose.

In Escherichia coli, the frequency of integration of a PCR-generated DNAfragment along with the short flanking region provided by theRSF-Red-TER plasmid is as high as the frequency obtained when using thepKD46 helper plasmid (Datsenko, K. A., Wanner, B. L., 2000, Proc. Natl.Acad. Sci. USA, 97, 6640-6645). However, expression of the λ Red genesis toxic to Pantoea ananatis. Cells transformed with the RSF-Red-TERhelper plasmid grow extremely slowly in LB medium containing IPTG(isopropyl-β-D-thiogalactopyranoside, 1 mM) and an appropriateantibiotic (25 μg/ml of chloramphenicol or 40 μg/ml of kanamycin), andthe efficiency of λ Red-mediated recombination is extremely low (10⁻⁸),if observed at all.

A variant strain of Pantoea ananatis which is resistant to expression ofall three of the λ Red genes was selected. For this purpose, theRSF-Red-TER plasmid was introduced into the Pantoea ananatis SC17 strain(U.S. Pat. No. 6,596,517) by electroporation. After an 18-hour culture,about 10⁶ transformants were obtained, and among these, 10 clones formedcolonies of a large size, and the remainder formed extremely smallcolonies. After an 18 hour culture, the large colonies were about 2 mm,and the small colonies were about 0.2 mm. While the small colonies didnot grow any more even when the culture was extended up to 24 hours, thelarge colonies continued to grow. One of the large colony Pantoeaananatis mutant strains which was resistant to expression of all threeof the λ Red genes (gam, bet, and exo) was used for further analysis.

The RSF-Red-TER plasmid DNA was isolated from one clone of the largecolony clones, and from several clones of the small colony clones, andtransformed again into Escherichia coli MG1655 to examine the ability ofthe plasmid to synthesize an active Red gene product. A controlexperiment for Red-dependent integration in the obtained transformantswas used to demonstrate that only the plasmid isolated from the largecolony clone induced expression of the λ Red genes required for theRed-dependent integration. In order to investigate whether theRed-mediated integration occurs in the selected large colony clone,electroporation was performed using a linear DNA fragment produced byPCR. This fragment was designed so that it contains a Km^(R) marker anda flanking region of 40 bp homologous to the hisD gene, and isintegrated into the hisD gene of Pantoea ananatis at the SmaIrecognition site. Two small colony clones were used as control. Thenucleotide sequence of the hisD gene of Pantoea ananatis is shown in SEQID NO: 40. For PCR, the oligonucleotides of SEQ ID NOS: 41 and 42 wereused as primers, and the pMW118-(λatt-Km^(r)-λatt) plasmid was used asthe template. The two small colony clones which were not resistant tothe λ Red genes were used as the control. Construction of thepMW118-(λattL-Kmr-λattR) plasmid will be explained in detail inReference Example 3.

The RSF-Red-TER plasmid can induce expression of the Red genes by thelacI gene carried on the plasmid. Two kinds of induction conditions wereinvestigated. In the first group, IPTG (1 mM) was added 1 hour beforethe electroporation, and in the second group, IPTG was added at thestart of the culture to prepare cells in which electroporation ispossible. The growth rate of the progeny of the SC17 strain harboringRSF-Red-TER derived from the large colony clone was not significantlylower than that of a strain not having the RSF-Red-TER plasmid. Theaddition of IPTG only slightly decreased the growth rate of thesecultures. On the other hand, the RSF-Red-TER-introduced SC17 strainderived from the progeny of the small colony clones grew extremelyslowly even without the addition of IPTG, and after induction, growthwas substantially arrested. After electroporation of RSF-Red-TERisolated from the cells of the progeny of the large colony clone, manyKm^(R) clones grew (18 clones after a short induction time, and about100 clones after an extended induction time). All of the 100 clones thatwere investigated had a His phenotype, and about 20 clones wereconfirmed by PCR to have the expected structure of the chromosome in thecells. On the other hand, even when electroporation was performed withRSF-Red-TER isolated from cells of the progeny of the small colonyclones, an integrated strain was not obtained.

The large colony clone was grown on a plate containing 7% sucrose toeliminate the plasmid, and transformed again with RSF-Red-TER. Thestrain without the plasmid was designated SC17(0). This strain wasdeposited at the Russian National Collection of IndustrialMicroorganisms (VKPM), GNII Genetica (Address: 1 Dorozhny proezd., 1Moscow 117545, Russia) on Sep. 21, 2005, and assigned an accessionnumber of VKPM B-9246.

All the clones which grew after the aforementioned re-transformationwere large like the parent strain clone SC17(0). The Red-mediatedintegration experiment was performed in the SC17(0) strain which hadbeen re-transformed with the RSF-Red-TER plasmid. Three of theindependent transformants obtained were investigated using the same DNAfragment as that used for the previous experiment. A short inductiontime (1 hour before electroporation) was employed. Km^(R) clonesexceeding ten clones grew in each experiment. All the examined cloneshad the His phenotype. In this way, a mutant strain designated SC17(0)which is resistant to the expression of the λ Red genes was selected.This strain can be used as a recipient strain suitable for theRed-dependent integration into the Pantoea ananatis chromosome.

Reference Example 2 Construction of Helper Plasmid RSF-Red-TER

The scheme for constructing the helper plasmid RSF-Red-TER is shown inFIG. 2.

As the first step in the construction, an RSFsacBPlacMCS vector wasdesigned. For this purpose, DNA fragments containing the cat gene of thepACYC184 plasmid and the structural region of the sacB gene of Bacillussubtilis were amplified by PCR using the oligonucleotides of SEQ ID NOS:43 and 44, and 45 and 46, respectively. These oligonucleotides containedthe BglII, SacI, XbaI and BamHI restriction enzyme sites in the 5′ endregions. These sites are required and convenient for further cloning.The obtained sacB fragment of 1.5 kb was cloned into the previouslyobtained pMW119-P_(lac)lacI vector at the XbaI-BamHI site. This vectorwas constructed in the same manner as that described for thepMW118-P_(lac)lacI vector (Skorokhodova, A. Y. et al, 2004,Biotekhnologiya (Rus), 5:3-21). However, this vector contained apolylinker moiety derived from pMW219 instead of the pMW218 plasmid.

Then, the aforementioned cat fragment of 1.0 kb was treated with BglIIand SacI, and cloned into the RSF-P_(lac)lacIsacB plasmid obtained inthe previous step at the BamHI-SacI site. The obtained plasmidpMW-P_(lac)lacIsacBcat contained the PlacUV5-lacI-sacB-cat fragment. Inorder to subclone this fragment into the RSF1010 vector,pMW-P_(lac)lacIsacBcat was digested with BglII, blunt-ended with DNApolymerase I Klenow fragment, and successively digested with SacI. A 3.8kb BglII-SacI fragment of the pMWP_(lac)lacIsacBcat plasmid was elutedfrom a 1% agarose gel, and ligated with the RSF1010 vector which hadbeen treated with PstI and SacI. Escherichia coli TG1 was transformedwith the ligation mixture, and plated on the LB medium containingchloramphenicol (50 mg/L). The plasmids isolated from the grown cloneswere analyzed with restriction enzymes to obtain an RSFsacB plasmid. Inorder to construct an RSFsacBPlacMCS vector, a DNA fragment containingthe PlacUV5 promoter was amplified by PCR using the oligonucleotides ofSEQ ID NOS: 47 and 48 as primers and the pMW119-P_(lac)lacI plasmid as atemplate. The obtained fragment of 146 bp was digested with SacI andNotI, and ligated with the SacI-NotI large fragment of the RSFsacBplasmid. Then, by PCR using the oligonucleotides of SEQ ID NOS: 49 and50 as primers, and the pKD46 plasmid (Datsenko, K. A., Wanner, B. L.,2000, Proc. Natl. Acad. Sci. USA, 97, 6640-6645) as a template, a DNAfragment of 2.3 kb containing the λRedαβγ genes and the transcriptionterminator tL3 was amplified. The obtained fragment was cloned into theRSFsacBPlacMCS vector at the PvuI-NotI site. In this way, the RSFRedplasmid was designed.

In order to eliminate read through transcription of the Red genes, aρ-dependent transcription terminator of the rrnB operon of Escherichiacoli was inserted at a position between the cat gene and the PlacUV5promoter. For this purpose, a DNA fragment containing the PlacUV5promoter and the TrrnB terminator was amplified by PCR using theoligonucleotides of SEQ ID NOS: 51 and 48 as primers and the chromosomeof Escherichia coli BW3350 as the template. These obtained fragmentswere treated with KpnI and ligated. Then, the 0.5 kb fragment containingboth PlacUV5 and TrrnB was amplified by PCR using the oligonucleotidesof SEQ ID NOS: 48 and 52 as primers. The obtained DNA fragment wasdigested with EcoRI, blunt-ended by a treatment with DNA polymerase IKlenow fragment, digested with BamHI, and ligated with theEc1136II-BamHI large fragment of the RSFsacBPlacMCS vector. The obtainedplasmid was designated RSF-Red-TER.

Reference Example 3 Construction of the pMW118-(λattL-Km^(r)-λattR)Plasmid

The pMW118-(λattL-Km^(r)-λattR) plasmid was constructed from thepMW118-attL-Tc-attR (WO2005/010175) plasmid by replacing thetetracycline resistance marker gene with the kanamycin resistance geneof the pUC4K plasmid. For that purpose, the EcoRI-HindIII large fragmentfrom pMW118-attL-Tc-attR was ligated to two fragments from the pUC4Kplasmid: the HindIII-PstI fragment (676 bp) and EcoRI-HindIII fragment(585 bp). Basic pMW118-attL-Tc-attR was obtained by ligation of thefollowing four fragments.

1) The BglII-EcoRI fragment (114 bp) including attL (SEQ ID NO: 55)which was obtained by PCR amplification of the region corresponding toattL of the Escherichia coli W3350 (containing λ prophage) chromosomeusing the primers P1 and P2 (SEQ ID NOS: 53 and 54) (these primerscontained the subsidiary recognition sites for BglII and EcoRI).

2) The PstI-HindIII fragment (182 bp) including attR (SEQ ID NO: 58)which was obtained by PCR amplification of the region corresponding toattR of the Escherichia coli W3350 (containing λ prophage) chromosomeusing the primers P3 and P4 (SEQ ID NOS: 56 and 57) (these primerscontained the subsidiary recognition sites for PstI and HindIll).

3) The BglII-HindIII large fragment (3916 bp) of pMW118-ter_rrnB. Theplasmid pMW118-ter_rrnB was obtained by ligation of the following threeDNA fragments:

The large DNA fragment (2359 bp) including the AatII-EcoRI fragment ofpMW118 that was obtained by digesting pMW118 with EcoRI, treated withDNA polymerase I Klenow fragment, and then digested with AatII;

The small AatII-BglII fragment (1194 bp) of pUC19 including the bla genefor ampicillin resistance (ApR), which was obtained by PCR amplificationof the corresponding region of the pUC19 plasmid using the primers P5and P6 (SEQ ID NOS: 59 and 60) (these primers contained the subsidiaryrecognition sites for PstI, AatII and BglII);

The small BglII-PstI fragment (363 bp) of the transcription terminatorter_rrnB, which was obtained by PCR amplification of the correspondingregion of the Escherichia coli MG1655 chromosome using the primers P7and P8 (SEQ ID NOS: 61 and 62) (these primers contained the subsidiaryrecognition sites for PstI, BglII and PstI).

4) The small EcoRI-PstI fragment (1388 bp) (SEQ ID NO: 63) ofpML-Tc-ter_thrL including the tetracycline resistance gene and theter_thrL transcription terminator; the pML-Tc-ter_thrL plasmid wasobtained by the following two steps:

the pML-ter_thrL plasmid was obtained by digesting the pML-MCS plasmid(Mashko, S. V. et al., 2001, Biotekhnologiya (in Russian), no. 5, 3-20)with XbaI and BamHI, followed by ligation of the large fragment (3342bp) with the XbaI-BamHI fragment (68 bp) carrying ter_thrL terminatorobtained by PCR amplification of the corresponding region of theEscherichia coli MG1655 chromosome using the primers P9 and P10 (SEQ IDNOS: 64 and 65) (these primers contained the subsidiary recognitionsites for PstI, XbaI and BamHI);

the pML-Tc-ter_thrL plasmid was obtained by digesting the pML-ter_thrLplasmid with KpnI and XbaI followed by treatment with Klenow fragment ofDNA polymerase I and ligated with the small EcoRI-Van91I fragment (1317bp) of pBR322 including the tetracycline resistance gene (pBR322 wasdigested with EcoRI and Van91I and then treated with DNA polymerase IKlenow fragment).

Example 1 Search of L-Glutamic Acid Secretion Gene

A search for an L-glutamic acid secretion gene was performed as follows.Since L-glutamic acid is converted into an intermediate of thetricarboxylic acid cycle, 2-oxoglutarate, in one step by glutamatedehydrogenase, L-glutamic acid is thought to be easily metabolized inmany microorganisms having glutamate dehydrogenase or the tricarboxylicacid cycle. However, since a strain in which 2-oxoglutaratedehydrogenase is deleted cannot degrade L-glutamic acid, growth of thecells is inhibited in the presence of a high concentration glutamicacid. In this example, the SC17sucAams strain derived from the Pantoeaananatis SC17sucA strain (refer to Japanese Patent Laid-open No.2001-333769) is deficient in the extracellular polysaccharidebiosynthesis system, and is also deficient in 2-oxoglutaratedehydrogenase. Therefore, this strain was used to try to obtain anL-glutamic acid excretion gene utilizing resistance to a highconcentration of L-glutamic acid as a marker.

Since the SC17sucA strain produced a marked amount of extracelluarpolysaccharides when grown on an agar medium containing a sugar source,the handling of this strain is extremely difficult. Therefore, bydeleting the ams operon coding for the extracellular polysaccharidebiosynthesis system genes, production of extracellular polysaccharideswas suppressed. PCR was performed by using pMW118-λattL-Km^(r)-λattR asthe template and the primers of SEQ ID NOS: 6 and 7 to amplify a genefragment containing a kanamycin resistance gene, attL and attR sequencesof λ phage at the both ends of the resistance gene, and 50 by upstreamsequence of amsI and 50 bp downstream sequence of the amsC gene added tothe outer ends of the λ phage sequences. This fragment was purified byusing Wizard PCR Prep DNA Purification System (Promega).

Then, the SC17(0) strain was transformed with RSF-Red-TER to obtain anSC17(0)/RSF-Red-TER strain. This strain was cultured overnight in Lmedium (10 g of Bacto tryptone, 5 g of yeast extract and 5 g of NaCl in1 L of pure water, pH 7.0) containing 25 mg/L of chloramphenicol, andthen the culture medium after the overnight culture was inoculated in1/100 volume into 100 mL of the L medium containing 25 mg/L ofchloramphenicol and 1 mM isopropyl-β-D-thiogalactopyranoside, andculture was performed at 34° C. for 3 hours. The cells prepared asdescribed above were collected, washed three times with ice-cooled 10%glycerol, and finally suspended in 0.5 mL of 10% glycerol. The suspendedcells were used as competent cells, and 100 ng of the PCR fragmentprepared in the above section was introduced into the cells by usingGENE PULSER II (BioRad) with a field strength of 18 kV/cm, capacitorcapacity of 25 μF and resistance of 200 Ω. Ice-cooled SOC medium (20 g/Lof Bacto tryptone, 5 g/L of yeast extract, 0.5 g/L of NaCl and 10 g/L ofglucose) was added to the cell suspension, and culture was performed at34° C. for 2 hours with shaking. The culture was applied to a mediumprepared by adding ingredients of minimal medium (5 g of glucose, 2 mMmagnesium sulfate, 3 g of monopotassium phosphate, 0.5 g of sodiumchloride, 1 g of ammonium chloride and 6 g of disodium phosphate in 1 L)and 40 mg/L of kanamycin to the L medium (10 g of Bacto tryptone, 5 g ofyeast extract, 5 g of NaCl and 15 g of agar in 1 L of pure water, pH7.0). The colonies that appeared were purified with the same medium, andthen it was confirmed by PCR that the ams gene had been replaced withthe kanamycin resistance gene.

The chromosome was extracted from the ams gene-deficient strain using aBacterial Genomic DNA Purification Kit (Edge Biosystems). Separately,the SC17sucA strain was cultured overnight on an agar medium obtained byadding the ingredients of the minimal medium described above to the Lmedium. The cells were scraped with a loop, washed three times withice-cooled 10% glycerol, and finally suspended in 10% glycerol to afinal volume of 500 μL. The suspended cells were used as competentcells, and 600 ng of the aforementioned chromosome DNA was introducedinto the competent cells using GENE PULSER II (BioRad) with a fieldstrength of 17.5 kV/cm, capacitor capacity of 25 μF and resistance of200 Ω. Ice-cooled SOC medium was added to the cell suspension, andculture was performed at 34° C. for 2 hours with shaking. Then, theculture was applied on an agar medium prepared by adding ingredients ofthe minimal medium described above and 40 mg/L of kanamycin to the Lmedium. The colonies that appeared were purified with the same medium,and then it was confirmed by PCR that the ams gene had been replacedwith the kanamycin resistance gene. This strain was designated asSC17sucAams.

Chromosomal DNA extracted from the Pantoea ananatis AJ13355 strain waspartially digested with the restriction enzyme Sau3AI. Then, fragmentsof about 10 kb were collected and introduced into the BamHI site ofpSTV28 (Takara Bio) to prepare a plasmid library. This plasmid librarywas introduced into competent cells of the SC17sucAams strain preparedin a conventional manner by electroporation.

Selection was performed for the SC17sucAams strain which had beenintroduced with a plasmid library on a plate of the L medium (10 g ofBacto tryptone, 5 g of yeast extract, 5 g of NaCl and 15 g of agar in 1L of pure water, pH 7.0) added with ingredients of minimal medium (5 gof glucose, 2 mM magnesium sulfate, 3 g of monopotassium phosphate, 0.5g of sodium chloride, 1 g of ammonium chloride and 6 g of disodiumphosphate in 1 L of pure water) using chloramphenicol resistance as amarker to obtain transformants. These transformants were plated on aglucose minimal medium containing a high concentration L-glutamic acid(the glucose minimal medium mixed with 0.2 M L-glutamic acid, 100 mg/Leach of lysine, methionine and diaminopimelic acid as finalconcentrations), in which SC17sucAams cannot form colonies.

The transformants were cultured at 34° C. for 3 days, and 64 clonesamong the colonies that appeared were allowed to again form singlecolonies on the same plate. As a result, 11 clones were found to formcolonies after 48 hours, and the remaining colonies formed coloniesafter 72 hours.

Then, the genes inserted into the vectors harbored by the transformantswere analyzed. Plasmids were extracted from the transformants, andnucleotide sequences were determined. It was found that among the 11clones that formed colonies within 48 hours, 10 clones had the sameloci, and all contained genes showing homology to ybjL, which was anEscherichia coli gene of unknown function. In addition, this ybjL genecould not be obtained under the same conditions that the glutamic acidsecretion system gene described in WO2005/085419, yhfK, was obtained,and conversely, the yhfK gene could not be obtained under the selectionconditions used in this example.

In order to confirm that the factor which imparts glutamic acidresistance is ybjL, the ybjL gene was cloned. PCR was performed usingthe chromosomal DNA of AJ13355 strain as the template andoligonucleotides ybjL-F1 and ybjL-R2 shown in SEQ ID NOS: 8 and 9 toamplify the fragment of about 1.8 kb containing the ybjL gene of P.ananatis. This fragment was purified using Wizard PCR Prep DNAPurification System (Promega), and then treated with the restrictionenzymes KpnI and SphI, and the product was ligated with pSTV28 (TakaraBio) which had been treated with the same enzymes to obtainpSTV-PanybjL. The SC17sucA strain was transformed with this pSTV-PanybjLplasmid, and using pSTV28 as a control for comparison (Takara Bio) toconstruct the SC17sucA/pSTV-PanybjL and SC17sucA/pSTV28 strains.

The SC17sucA/pSTV-ybjL strain was plated on minimal medium (5 g ofglucose or sucrose, 2 mM magnesium sulfate, 3 g of monopotassiumphosphate, 0.5 g of sodium chloride, 1 g of ammonium chloride, 6 g ofdisodium phosphate, 0.2 M sodium L-glutamate, 100 mg/L each of lysine,methionine and diaminopimelic acid and 15 g of agar in 1 L of purewater) containing glutamic acid. Culture was performed at 34° C. for 2days. As a result, it was confirmed that whereas the controlvector-introduced strain, SC17sucA/pSTV28, could not form colonies,SC17sucA/pSTV-ybjL could form colonies on the minimal medium.

Then, the SC17sucA/pSTV-PanybjL strain was cultured in liquid minimalmedium (5 g of glucose, 2 mM magnesium sulfate, 3 g of monopotassiumphosphate, 0.5 g of sodium chloride, 1 g of ammonium chloride, 6 g ofdisodium phosphate, 0.2 M sodium L-glutamate, 100 mg/L each of lysine,methionine and diaminopimelic acid in 1 L of pure water) containingglutamic acid to examine growth of the strain in the presence of a highconcentration of L-glutamic acid.

The results are shown in FIG. 3. It was found that growth ofSC17sucA/pSTV-PanybjL in which the ybjL gene had been enhanced wasmarkedly improved in the presence of high concentration L-glutamic acidas compared to the control strain SC17sucA/pSTV28. Accordingly, it wasconfirmed that ybjL is a factor which imparts resistance to L-glutamicacid.

Example 2 Effect of ybjL Gene Amplification on L-Glutamic AcidProduction at Neutral pH

Then, in order to examine the effect of this gene on L-glutamic acidproduction, the plasmid for ybjL amplification, pSTV-PanybjL, wasintroduced into the L-glutamic acid producing bacterium SC17sucA/RSFCPGhaving the plasmid for L-glutamic acid production, RSFCPG, shown in SEQID NO: 10 (refer to Japanese Patent Laid-open No. 2001-333769), andL-glutamic acid productivity thereof was examined.

pSTV-PanybjL and the control plasmid, pSTV28 (Takara Bio), were eachintroduced into SC17sucA/RSFCPG by electroporation, and transformantswere obtained using chloramphenicol resistance as a marker. Afterconfirmation of the presence of the plasmids, the strain with theplasmid for ybjL amplification was designatedSC17sucA/RSFCPG+pSTV-PanybjL, and the strain with pSTV29 was designatedSC17sucA/RSFCPG+pSTV28.

Then, SC17sucA/RSFCPG+pSTV-PanybjL and the control strainSC17sucA/RSFCPG+pSTV28 were cultured to examine the L-glutamic acidproducing ability of the strains. The medium had the followingcomposition:

Composition of culture medium:

section A:

Sucrose  30 g/L MgSO₄ · 7H₂O 0.5 g/L

[[Group B:

KH₂PO₄  2.0 g/L Yeast Extract  2.0 g/L FeSO₄ · 7H₂O 0.02 g/L MnSO₄ ·5H₂O 0.02 g/L L-Lysine hydrochloride  0.2 g/L DL-Methionine  0.2 g/LDiaminopimelic acid  0.2 g/L (adjusted to pH 7.0 with KOH)

Group C:

CaCO₃ 20 g/L

The ingredients of groups A and B were sterilized at 115° C. for 10minutes by autoclaving, and the ingredient of group C was sterilized at180° C. for 3 hours with dry heat. Then, the ingredients of the threegroups were mixed, and 12.5 mg/L of tetracycline hydrochloride and 25mg/L of chloramphenicol were added to the mixture.

SC17sucA/RSFCPG+pSTV29 and SC17sucA/RSFCPG+pSTV-PanybjL were eachprecultured on a medium obtained by adding ingredients of the minimalmedium (medium containing 5 g of glucose, 2 mM magnesium sulfate, 3 g ofmonopotassium phosphate, 0.5 g of sodium chloride, 1 g of ammoniumchloride and 6 g of disodium phosphate in 1 L of pure water), 25 mg/L ofchloramphenicol and 12.5 mg/L tetracycline to the L medium (mediumcontaining 10 g of Bacto tryptone, 5 g of yeast extract, 5 g of NaCl and15 g of agar in 1 L of pure water, pH 7.0), and inoculated into anappropriate amount to 5 ml of the aforementioned medium in a test tube.

The cells were cultured for 17.5 hours, and then cell density,L-glutamic acid concentration, and the amount of residual saccharide inthe culture medium were measured. The cell density was examined bymeasuring turbidity at 620 nm of the medium diluted 51 times using aspectrophotometer (U-2000A, Hitachi). The L-glutamic acid concentrationwas measured in culture supernatant appropriately diluted with water byusing Biotech Analyzer (AS-210, Sakera SI). The results are shown inTable 1. L-Glutamic acid accumulation was increased by about 3 g/L, andthe yield based on saccharide increased by about 9% in theybjL-amplified strain, SC17sucA/RSFCPG+pSTV-PanybjL, as compared to thecontrol strain, SC 17sucA/RSFCPG+pSTV28.

TABLE 1 OD 620 nm L-Glutamic Yield based on (×51) acid (g/L) saccharide(%) SC17sucA/RSFCPG + 0.385 15.0 44.5 pSTV28 SC17sucA/RSFCPG + 0.28018.0 53.7 pSTV-PanybjL

Example 3 Effect of Amplification of the ybjL Gene Derived fromEscherichia coli

Then, the ybjL gene from Escherichia coli was introduced into thePantoea ananatis SC17sucA/RSFCPG strain, and the effect of thisamplification on glutamic acid production was examined.

PCR was performed using the oligonucleotides shown in SEQ ID NOS: 11 and12 prepared on the basis of the sequence of the ybjL of Escherichia coliregistered at GeneBank as AP009048 (SEQ ID NO: 3) and the chromosomefrom the Escherichia coli W3110 strain (ATCC 27325) as the template toobtain a fragment of about 1.7 kb containing the ybjL gene. Thisfragment was treated with SphI and KpnI, and ligated with pSTV28 (TakaraBio) at the corresponding site. The plasmid for amplification of ybjL ofEscherichia coli was designated as pSTV-EcoybjL.

The plasmid pSTV-EcoybjL for ybjL amplification was introduced into theaforementioned SC17sucA/RSFCPG strain by electroporation, and atransformant was obtained using chloramphenicol resistance as a marker.The obtained Escherichia coli ybjL gene-amplified strain was designatedas SC17sucA/RSFCPG+pSTV-EcoybjL.

Then, SC17sucA/RSFCPG+pSTV-EcoybjL and the control strain,SC17sucA/RSFCPG+pSTV28, were cultured to examine their L-glutamic acidproducing ability. The medium had the following composition.

Composition of culture medium:

Group A:

Sucrose  30 g/L MgSO₄ · 7H₂O 0.5 g/L

Group B:

KH₂PO₄  2.0 g/L Yeast Extract  2.0 g/L FeSO₄ · 7H₂O 0.02 g/L MnSO₄ ·5H₂O 0.02 g/L L-Lysine hydrochloride  0.2 g/L DL-Methionine  0.2 g/LDiaminopimelic acid  0.2 g/L (adjusted to pH 7.0 with KOH)

Group C:

CaCO₃ 20 g/L

The ingredients of groups A and B were sterilized at 115° C. for 10minutes by autoclaving, and the ingredient of group C was sterilized at180° C. for 3 hours with dry heat. Then, the ingredients of the threegroups were mixed, and 12.5 mg/L of tetracycline hydrochloride and 25mg/L of chloramphenicol were added to the mixture.

SC17sucA/RSFCPG+pSTV28 and SC17sucA/RSFCPG+pSTV-EcoybjL were eachprecultured on a medium obtained by adding ingredients of the minimalmedium (5 g of glucose, 2 mM magnesium sulfate, 3 g of monopotassiumphosphate, 0.5 g of sodium chloride, 1 g of ammonium chloride and 6 g ofdisodium phosphate in 1 L of pure water), 25 mg/L of chloramphenicol,and 12.5 mg/L tetracycline to the L medium (10 g of Bacto tryptone, 5 gof yeast extract, 5 g of NaCl and 15 g of agar in 1 L of pure water, pH7.0), and inoculated in an appropriate amount to 5 ml of theaforementioned medium in a test tube.

The cells were cultured for 17.5 hours, and then cell density andL-glutamic acid concentration in the culture medium were measured in thesame manner as shown in Example 2. The results are shown in Table 2.L-Glutamic acid accumulation was increased by about 2 g/L, and the yieldbased on saccharide increased by about 7% in the ybjL-amplified strain,SC17sucA/RSFCPG+pSTV-EcoybjL, as compared to the control strain,SC17sucA/RSFCPG+pSTV28.

TABLE 2 OD 620 nm L-Glutamic acid Yield based on (×51) (g/L) saccharide(%) SC17sucA/RSFCPG + 0.385 15.0 44.5 pSTV28 SC17sucA/RSFCPG + 0.34817.2 51.2 pSTV-EcoybjL

Example 4 Effect of Amplification of ybjL Gene on L-Amino AcidProduction in Escherichia coli

Then, the ybjL gene derived from Pantoea ananatis and the ybjL genederived from Escherichia coli were each introduced into Escherichiacoli, and the effect of the amplification was examined.

The aforementioned vector for amplification of the ybjL gene derivedfrom Pantoea ananatis, pSTV-PanybjL, vector for amplification of ybjLgene derived from Escherichia coli, pSTV-EcoybjL, and the controlplasmid pSTV28 were each introduced into an Escherichia coli wild-typestrain, W3110, by electroporation to obtain transformants resistant tochloramphenicol. The strain in which ybjL derived from Pantoea ananatishad been amplified was designated W3110/pSTV-PanybjL, the strain inwhich ybjL derived from Escherichia coli had been amplified wasdesignated W3110/pSTV-EcoybjL, and the control strain with pSTV28 wasdesignated W3110/pSTV28.

Then, the ability to produce L-glutamic acid of the ybjL-amplifiedstrains, W3110/pSTV-PanybjL and W3110/pSTV-EcoybjL, and the controlstrain W3110/pSTV28 were examined. W3110/pSTV-PanybjL,W3110/pSTV-EcoybjL, and W3110/pSTV28 were each precultured on L medium(10 g of Bacto tryptone, 5 g of yeast extract, 5 g of NaCl and 15 g ofagar in 1 L of pure water, pH 7.0) containing chloramphenicol, and oneloop of cells were inoculated by using a 1-mL volume loop (Nunc) to 5 mLof a medium having the following composition in a test tube, andcultured at 37° C. for 11.5 hours with shaking. Cell density andL-glutamic acid concentration in the culture medium were measured in thesame manners as those in Example 2. The results are shown in Table 3.

Composition of culture medium:

Group A:

Glucose 30 g/L MgSO₄•7H₂O 0.5 g/L

Group B:

(NH₄)₂SO₄ 20 g/L KH₂PO₄ 2.0 g/L Yeast Extract 2.0 g/L FeSO₄•7H₂O 20 mg/LMnSO₄•5H₂O 20 mg/L (adjusted to pH 7.0 with KOH)

Group C:

Calcium carbonate 20 g/L

The ingredients of groups A and B were sterilized at 115° C. for 10minutes by autoclaving, and the ingredient of group C was sterilized at180° C. for 3 hours with dry heat. Then, the ingredients of the threegroups were mixed, and 25 mg/L of chloramphenicol was added to themixture.

TABLE 3 OD 620 nm L-Glutamic Yield based on (×51) acid (g/L) saccharide(%) W3110/pSTV28 0.341 1.2 6.5 W3110/pSTV-PanybjL 0.303 5.7 28.8W3110/pSTV-EcoybjL 0.310 4.8 25.2

The L-glutamic acid producing ability was markedly improved in theEscherichia coli W3110/pSTV-PanybjL strain, which is a Pantoea ananatisin which the ybjL gene has been amplified, and the Escherichia coliW3110/pSTV-EcoybjL strain, which is an Escherichia coli in which theybjL gene has been amplified, as compared to the the control strainW3110/pSTV28 strain.

Then, the ability of the ybjL-amplified strain to produce other aminoacids was examined. W3110/pSTV-PanybjL, and the control strainW3110/pSTV28 were precultured in L medium (10 g of Bacto tryptone, 5 gof yeast extract, 5 g of NaCl and 15 g of agar in 1 L of pure water, pH7.0) containing chloramphenicol, and one loop of cells were inoculatedby using a 5-mL volume loop (Nunc) to 5 mL of a medium having thefollowing composition in a test tube, and cultured at 37° C. for 7 hourswith shaking. Cell densities and L-amino acid concentrations in theculture medium were measured in the same manner as in Example 2. Theresults are shown in Table 4.

Composition of culture medium:

Group A:

Glucose 40 g/L MgSO₄•7H₂O 0.5 g/L

Group B:

(NH₄)₂SO₄ 20 g/L KH₂PO₄ 2.0 g/L Yeast Extract 2.0 g/L FeSO₄•7H₂O 20 mg/LMnSO₄•5H₂O 20 mg/L (adjusted to pH 7.0 with KOH)

Group C:

Calcium carbonate 30 g/L

The ingredients of groups A and B were sterilized at 115° C. for 10minutes by autoclaving, and the ingredient of group C was sterilized at180° C. for 3 hours with dry heat. Then, the ingredients of the threegroups were mixed, and 25 mg/L of chloramphenicol was added to themixture.

TABLE 4 W3110/pSTV- W3110/pSTV28 PanybjL MS medium* Asp 26.69 62.3540.21 Thr 0.00 0.00 36.23 Ser 0.00 0.00 56.03 Glu 1258.75 3702.74 127.50Gly 0.00 0.00 27.28 Ala 5.49 9.79 77.61 (Cys)₂ 16.49 13.72 10.13 Val3.30 8.02 59.32 Met 0.00 0.00 85.06 Ile 0.00 0.00 50.51 Leu 0.00 0.0080.02 Tyr 48.20 0.00 38.80 Phe 2.88 4.47 70.49 Lys 8.07 8.00 50.50 His0.00 0.00 20.95 Arg 0.00 0.00 35.80 OD 620 nm (×51) 0.245 0.197 —Residual glucose 28.9 28.3 40.0 (g/L) *Blank inoculated with no cells

Not only did the amount of L-glutamic acid increase, but the L-asparticacid amount also increased in the Escherichia coli W3110/pSTV-PanybjL,which is a ybjL gene-amplified strain, as compared to thevector-introduced control strain, W3110/pSTV28 strain.

Example 5 Effect of Amplification of the ybjL Gene on L-Glutamic AcidProduction in Klebsiella planticola

The ybjL gene derived from Pantoea ananatis was introduced intoKlebsiella planticola, and the effect of the amplification of the genewas examined.

The aforementioned vector for amplification of ybjL gene derived fromPantoea ananatis, pSTV-PanybjL, and the control plasmid pSTV28 were eachintroduced into a Klebsiella planticola L-glutamic acid-producingstrain, AJ13410 (Japanese Patent Application No. 11-68324), byelectroporation to obtain transformants which are resistant tochloramphenicol. The strain in which ybjL derived from Pantoea ananatishad been amplified was designated as AJ13410/pSTV-PanybjL, and thecontrol strain with pSTV28 was designated as AJ13410/pSTV28.

Then, the ability to produce L-glutamic acid of the ybjL-amplifiedstrain, AJ13410/pSTV-PanybjL, and the control strain AJ13410/pSTV28 wereexamined. AJ13410/pSTV-PanybjL and AJ13410/pSTV28 were precultured on Lmedium (10 g of Bacto tryptone, 5 g of yeast extract, 5 g of NaCl and 15g of agar in 1 L of pure water, pH 7.0) containing chloramphenicol, andone loop of cells were inoculated by using a 1-mL volume loop (Nunc) to5 mL of a medium having the following composition in a test tube, andcultured at 37° C. for 17 hours with shaking. Cell density andL-glutamic acid concentration in the culture medium were measured in thesame manner as in Example 2. The results are shown in Table 5.

Composition of culture medium:

Group A:

Sucrose 30 g/L MgSO₄•7H₂O 0.5 g/L

Group B:

KH₂PO₄ 2.0 g/L Yeast Extract 2.0 g/L FeSO₄•7H₂O 0.02 g/L MnSO₄•5H₂O 0.02g/L L-Lysine hydrochloride 0.2 g/L DL-Methionine 0.2 g/L Diaminopimelicacid 0.2 g/L (adjusted to pH 7.0 with KOH)

Group C:

CaCO₃ 20 g/L

The ingredients of groups A and B were sterilized at 115° C. for 10minutes by autoclaving, and the ingredient of group C was sterilized at180° C. for 3 hours with dry heat. Then, the ingredients of the threegroups were mixed, and 25 mg/L of chloramphenicol was added to themixture.

TABLE 5 OD 620 nm L-Glutamic Yield based on (×51) acid (g/L) saccharide(%) AJ13410/pSTV28 0.236 5.3 23.3 AJ13410/pSTV-PanybjL 0.220 12.1 48.7

L-glutamic acid-producing ability was markedly improved in theKlebsiella planticola AJ13410/pSTV-PanybjL, which is a Pantoea ananatisybjL gene-amplified strain, as compared to the control strain,AJ13410/pSTV28.

Reference Example 4 Construction of an Escherichia coli Strain Deficientin the L,D-Lactate Dehydrogenase Gene

This gene was deleted by using the method called “Red-drivenintegration” developed by Datsenko and Wanner (Datsenko, K. A. andWanner, B. L., 2000, Proc. Natl. Acad. Sci. USA, vol. 97, No. 12, pp.6640-6645), and the λ phage excision system (Cho, E. H. Gumport, R. I.and Gardner, J. F., 2002, J. Bacteriol., 184 (18):5200-5203). Accordingto this method, it is possible, to construct a gene-disrupted strain ina single step by using a PCR product obtained with syntheticoligonucleotide primers in which a part of the target gene is designedin the 5′ end and a part of an antibiotic resistance gene is designed inthe 3′ end. Furthermore, by using the λ phage excision system incombination, the antibiotic resistance gene which is integrated into thegene-disrupted strain can be removed.

<1-1> Construction of a Strain Deficient in the ldhA Gene Coding forD-Lactate Dehydrogenase

According to the description of WO2005/010175, PCR was performed byusing synthetic oligonucleotides having sequences corresponding to partsof the ldhA gene in the 5′ end and sequences corresponding to both endsof attL and attR of λ phage at the 3′ end as primers and plasmidpMW118-attL-Cm-attR as the template. pMW118-attL-Cm-attR is a plasmidobtained by inserting attL and attR genes, which are the attachmentsites for λ phage, and the cat gene, which is an antibiotic resistancegene, into pMW118 (Takara Bio), and the genes are inserted in the orderof attL-cat-attR. The sequences of the synthetic oligonucleotides usedas the primers are shown in SEQ ID Nos. 13 and 14. The amplified PCRproduct was purified on an agarose gel and introduced into Escherichiacoli MG1655 strain containing the plasmid pKD46, which is capable oftemperature-sensitive replication by eletroporation. Then, anampicillin-sensitive strain not harboring pKD46 was obtained, and thedeletion of the ldhA gene was confirmed by PCR. PCR was performed byusing the synthetic oligonucleotides shown in SEQ ID NOS: 15 and 16 asprimers. Whereas the PCR product obtained for the parent strain wasabout 1.2 kb, the deficient strain was about 1.9 kb.

To eliminate the att-cat gene introduced into the ldhA gene, the strainwas transformed with helper plasmid pMW-intxis-ts, and an ampicillinresistant strain was selected. The pMW-intxis-ts contains the λ phageintegrase (Int) gene and excisionase (Xis) gene and showstemperature-sensitive replication. Then, the strain in which att-cat andpMW-intxis-ts had been eliminated was confirmed by PCR on the basis ofampicillin sensitivity and chloramphenicol sensitivity. PCR wasperformed by using the synthetic oligonucleotides shown in SEQ NOS: 15and 16 as primers. Whereas the PCR product obtained for the strain inwhich att-cat remained was about 1.9 kb, a band of about 0.3 kb wasobserved for the strain in which att-cat was eliminated. The ldhAdeficient strain obtained as described above was designated asMG1655ΔldhA strain.

<1-2> Construction of a Strain Deficient in the lldD Gene Coding forL-Lactate Dehydrogenase

A strain was constructed in the same manner as that of the constructionof the ldhA gene-deficient strain. PCR was performed using syntheticoligonucleotides having sequences corresponding to parts of the lldDgene in the 5′ end and sequences corresponding to both ends of attL andattR of λ phage in the 3′ end as primers and plasmid pMW118-attL-Cm-attRas the template. The sequences of the synthetic oligonucleotides used asthe primers are shown in SEQ ID Nos. 17 and 18. The amplified PCRproduct was purified on an agarose gel and introduced into theEscherichia coli MG1655ΔldhA strain containing plasmid pKD46 capable oftemperature-sensitive replication by eletroporation. Then, anampicillin-sensitive strain not harboring pKD46 was obtained, and thedeletion of the llD gene was confirmed by PCR. PCR was performed byusing the synthetic oligonucleotides shown in SEQ ID NOS: 19 and 20 asprimers. Whereas the PCR product obtained for the parent strain wasabout 1.4 kb, a band of about 1.9 kb was observed for the deficientstrain.

To eliminate the att-cat gene introduced into the lldD gene, the strainwas transformed with helper plasmid pMW-intxis-ts, and an ampicillinresistant strain was selected. Then, the strain from which att-cat andpiMW-intxis-ts had been eliminated was obtained and confirmed by PCR onthe basis of ampicillin sensitivity and chloramphenicol sensitivity. PCRwas performed by using the synthetic oligonucleotides shown in SEQ IDNOS: 19 and 20 as primers. Whereas the PCR product obtained for thestrain in which att-cat remained was about 1.9 kb, a band of about 0.3kb was observed for the strain in which att-cat was eliminated. The lldDdeficient strain obtained as described above was designated asMG1655ΔldhΔlldD strain.

Example 6 Construction of ybjL Gene-Enhanced Strain of SuccinicAcid-Producing Bacterium

<6-1> Construction of Plasmid for Gene Amplification

In order to amplify the ybjL gene, plasmid pMW219::Pthr was used. Thisplasmid corresponds to the vector pMW219 (Nippon Gene) having thepromoter region of the threonine operon (thrLABC) in the genome of theEscherichia coli shown in SEQ ID NO: 21 at the HindIII site and BamHisite, and enables amplification of a gene by cloning the gene at aposition in the plasmid downstream from that promoter.

<6-2> Construction of Plasmid for Enhancing ybjL

PCR was performed by using the synthetic oligonucleotide having a BamHIsite shown in SEQ ID NO: 22 as a 5′ primer, and the syntheticoligonucleotide having a BamHI site shown in SEQ ID NO: 23 as a 3′primer, which were prepared on the basis of the nucleotide, sequence ofthe ybjL gene in the genome sequence of Escherichia coli (GenBankAccession No. U00096), and the genomic DNA of Escherichia coli MG1655strain as the template. The product was treated with the restrictionenzyme BamHI to obtain a gene fragment containing the ybjL gene. The PCRproduct was purified and ligated with the vector pMW219::Pthr which hadbeen treated with BamHI to construct plasmid pMW219::Pthr::ybjL for ybjLamplification.

<6-3> Production of ybjL-Amplified Strain

pMW219::Pthr::ybjL obtained in <6-2> described above and pMW219 wereused to transform the Escherichia coli MG1655ΔldhAΔlldD strain by theelectric pulse method, and the transformants were applied to the LB agarmedium containing 25 μg/ml of kanamycin, and cultured at 37° C. forabout 18 hours. The colonies which appeared were purified, and plasmidswere extracted from them in a conventional manner to confirmintroduction of the target plasmid. The obtained strains were designatedas MG1655ΔldhAΔlldD/pMW219::Pthr::ybjL and MG1655ΔldhAΔlldD/pMW219respectively. The Enterobacter aerogenes AJ 110637 strain was alsotransformed with pMW219::Pthr::ybjL, and pMW219 by the electric pulsemethod, and the transformants were applied to the LB agar mediumcontaining 50 μg/ml of kanamycin, and cultured at 37° C. for about 18hours. The colonies which appeared were purified, and plasmids wereextracted from them in a conventional manner to confirm introduction ofthe target plasmid. The obtained strains were designated asAJ110637/pMW219::Pthr::ybjL and AJ110637/pMW219, respectively,

Example 7 Effect of ybjL Amplification in Succinic Acid-Producing Strainof Escherichia Bacterium

MG1655ΔldhAΔlldD/pMW219::Pthr::ybjL and MG1655ΔldhAΔlldD/pMW219 wereeach uniformly applied to an LB plate containing 25 μg/ml of kanamycin,and cultured at 37° C. for 16 hours. Then, each plate was incubated at37° C. for 16 hours under an anaerobic condition by using Anaeropack(Mitsubishi Gas Chemical). The cells on the plate were washed with 0.8%brine, and then diluted 51 times, and thereby a cell suspension havingOD=1.0 (600 nm) was prepared. This cell suspension in a volume of 100 μland a production medium (10 g/l of glucose, 10 g/l 2Na malate, 45.88 g/lof TES, 6 g/l of Na₂HPO₄, 3 g/l of KH₂PO₄, 1 g/l of NH₄Cl, adjusted topH 7.3 with KOH and filtered) in a volume of 1.3 ml in which thedissolved gases in the medium were replaced with nitrogen gas bybubbling nitrogen gas beforehand were put into 5-ml volume micro tubes,and the cells were cultured at 31.5° C. for 10 hours by using a stirrerfor micro tubes. After the culture, the amount of succinic acid whichhad accumulated in the medium was analyzed by liquid chromatography. TwoShim-pack SCR.-102H (Shimadzu) connected in series were used as thecolumn, and a sample was eluted at 50° C. with 5 mM p-toluenesulfonicacid. The eluate was neutralized with 20 mM Bis-Tris aqueous solutioncontaining 5 mM p-toluenesulfonic acid and 100 μM EDTA, and succinicacid was quantified by measuring electric conductivity with CDD-10AD(Shimadzu).

The amounts of succinic acid Which had accumulated after 10 hours areshown in Table 6, and the change in succinic acid accumulation is shownin FIG. 4.

Succinic acid accumulation was markedly increased in the ybjLgene-amplified strain MG1655ΔldhAΔlldD/pMW219::Pthr::ybjL as compared tothe control strain MG1655ΔldhAΔlldD/pMW219.

TABLE 6 Effect of ybjL amplification in succinic acid-producing strain,MG1655ΔldhAΔlldD Strain Succinate (g/L) MG1655ΔldhAΔlldD/pMW219 1.91(±0.13) MG1655ΔldhAΔlldD/pMW219::Pthr::ybjL 2.34 (±0.01)

Example 8 Effect of ybjL Amplification in a Succinic Acid-ProducingStrain of Enterobacter Bacterium

AJ110637/pMW219::Pthr::ybjL and AJ110637/pMW219 were each uniformlyapplied to an LB plate containing 50 μg/ml of kanamycin, and cultured at37° C. for 16 hours. Then, each plate was incubated at 37° C. for 16hours under an anaerobic condition by using Anaeropack (Mitsubishi GasChemical). The cells on the plate were washed with 0.8% brine, and thendiluted 51 times, and thereby a cell suspension having OD=1.0 (60 nm)was prepared. This cell suspension in a volume of 100 μl and aproduction medium (10 g/l of glucose, 10 g/l 2Na malate, 45.88 g/l ofTES, 6 g/l of Na₂HPO₄, 3 g/l of KH₂PO₄, 1 g/l of NH₄Cl, adjusted to pH7.3 with KOH and filtered) in a volume of 1.3 ml in which the dissolvedgases in the medium were replaced with nitrogen gas by bubbling nitrogengas beforehand were put into 5-ml volume micro tubes, and the cells werecultured at 31.5° C. for 6 hours by using a stirrer for micro tubes.After the culture, the amount of succinic acid which had accumulated inthe medium was analyzed by liquid chromatography. Two Shim-pack SCR-102HShimadzu) connected in series were used as the column, and a sample waseluted at 50° C. with 5 mM p-toluenesulfonic acid. The eluate wasneutralized with 20 mM Bis-Tris aqueous solution containing 5 mMp-toluenesulfonic acid and 100 μM EDTA, and succinic acid was quantifiedby measuring electric conductivity with CDD-10AD (Shimadzu).

The amounts of succinic acid which had accumulated after 6 hours areshown in Table 7, and change of succinic acid accumulation is shown inFIG. 5.

Succinic acid accumulation was markedly increased in the ybjLgene-amplified strain AJ110637/pMW219::Pthr::ybjL as compared to thecontrol strain AJ110637/pMW219.

TABLE 7 Effect of ybjL amplification in AJ110637 strain Strain Succinate(g/L) AJ110637/pMW219 1.81 (±0.04) AJ110637/pMW219::Pthr::ybjL 2.11(±0.02)

Example 9

<9-1> Construction of an adhE-Deficient Strain of Enterobacter aerogenesAJ110637

When Enterobacter aerogenes AJ110637 is grown in a medium containing asugar source under anaerobic conditions, it produces a marked amount ofethanol. Therefore, adhE coding for alcohol dehydrogenase was deleted tosuppress the generation of ethanol.

A gene fragment for deleting adhE was prepared by PCR using the plasmidpMW-attL-Tc-attR described in WO2005/010175 as the template andoligonucleotides of SEQ ID NOS: 72 and 73 as primers,pMW118-attL-Tc-attR is a plasmid obtained by inserting attL and attRgenes, which are the attachment sites of λ phage, and the Tc gene, whichis a tetracycline resistance gene, into pMW118 (Takara Bio), and thegenes are inserted in the order of attL-Tc-attR. By PCR described above,a gene fragment containing a tetracycline resistance gene, attL and attRsites of λ phage at the both ends of the resistance gene, and 60 bpupstream sequence and 59 bp downstream sequence of the adhE gene addedto the outer ends of the λ phage sequences was amplified. This fragmentwas purified by using Wizard PCR Prep DNA Purification System (Promega).

Then, the Enterobacter aerogenes AJ110637 strain was transformed withRSF-Red-TER to obtain the Enterobacter aerogenes AJ110637/RSF-Red-TERstrain. This strain was cultured overnight in LB medium containing 40μg/mL of chloramphenicol, the culture medium was inoculated in a 1/100volume to 50 mL of the LB medium containing 40 μg/mL of chloramphenicoland 0.4 mM isopropyl-β-D-thiogalactopyranoside, and culture wasperformed at 31° C. for 4 hours. The cells were collected, washed threetimes with ice-cooled 10% glycerol, and finally suspended in 0.5 mL of10% glycerol. The suspended cells were used as competent cells, and thePCR fragment prepared in the above section was introduced into the cellsby using GENE PULSER II (BioRad) under the conditions of a fieldstrength of 20 kV/cm, capacitor capacity of 25 μF and resistance of 200Ω. Ice-cooled LB medium was added to the cell suspension, and culturewas performed at 31° C. for 2 hours with shaking. Then, the culture wasapplied to a LB plate containing 25 μg/mL of tetracycline. The coloniesthat appeared were purified with the same plate, and then it wasconfirmed by PCR that the adhE gene had been replaced with thetetracycline resistance gene.

<9-2> Construction of Pyruvate Carboxylase Gene-Enhanced Strain ofAJ110637ΔadhE Strain

The ability to produce succinic acid was imparted to the Enterobacteraerogenes AJ110637ΔadhE strain by amplifying pyc coding for pyruvatecarboxylase derived from Corynebacterium glutamicum.

In order to express pyc derived from Corynebacterium glutamicum in theAJ110637ΔadhE strain, it was attempted to obtain a threonine operonpromoter fragment of the Escherichia coli MG1655 strain. The totalgenomic sequence of Escherichia coli (Escherichia coli K-12 strain) hasalready been elucidated (Genbank Accession No. U00096, Science, 277,1453-4474 (1997)). On the basis of this sequence, PCR amplification ofthe promoter region of the threonine operon (thrLABC) was performed. PCRwas performed by using the synthetic oligonucleotide having an SacI siteshown n SEQ ID NO: 75 as a 5′ primer, the synthetic oligonucleotideshown in SEQ ID NO: 76 as a 3′ primer, and the genomic DNA ofEscherichia coli MG1655 strain (ATCC 47076, ATCC 700926) as the templateto obtain a threonine operon promoter fragment (A) (SEQ ID NO: 77).

Furthermore, a pyc gene fragment derived from the Corynebacteriumglutamicum 2256 strain (ATCC 13869) was obtained. PCR was performed byusing the synthetic oligonucleotide shown in SEQ ID NO: 78 as a 5′primer, the synthetic oligonucleotide having an SacI site shown SEQ IDNO: 79 as a 3′ primer, and the genomic DNA of the Corynebacteriumglutamicum 2256 strain (ATCC 13869) as a template to obtain a pyc genefragment (B) (SEQ ID NO: 80).

PCR was performed by using the fragments (A) and (B) as templates, theprimers of SEQ ID NOS: 75 and 79 to obtain a gene fragment (C) includingthe fragments (A) and (B) ligated to each other. This gene fragment (C)was treated with the restriction enzyme SacI, and purified, and theproduct was ligated with the plasmid vector pSTV28 (Takara Bio) whichhad been digested with the restriction enzyme SacI to construct aplasmid pSTV28::Pthr::pyc for pyc amplification.

The plasmid pSTV28::Pthr::pyc for pyc amplification was introduced intothe aforementioned Enterobacter aerogenes AJ110637ΔadhE strain byelectroporation to obtain a transformant which are resistant totetracycline and chloramphenicol. This pyc-amplified strain ofEnterobacter aerogenes AJ110637ΔadhE was designated asAJ110637ΔadhE/pSTV28::Pthr::pyc.

<9-3> Construction of Enterobacter aerogenes ybjL Gene-Enhanced Strainof AJ110637ΔadhE/pSTV28::Pthr:pyc

In the same manner as that described above, PCR amplification of thepromoter region of the threonine operon (thrLABC) of Escherichia coli(Escherichia coli K-12 strain) was performed. PCR was performed by usingthe synthetic oligonucleotide shown in SEQ ID NO: 81 as a 5′ primer, thesynthetic oligonucleotide shown in SEQ ID NO: 82 as a 3′ primer, and thegenomic DNA of Escherichia coli MG1655 strain (ATCC 47076, ATCC 700926)as a template to obtain a threonine operon promoter fragment (A) (SEQ IDNO: 83).

Furthermore, in order to clone the ybjL gene of the Enterobacteraerogenes AJ110637 strain, PCR was performed by using the syntheticoligonucleotide shown in SEQ ID NO: 84 as a 5′ primer, the syntheticoligonucleotide shown in SEQ ID NO: 85 as a 3′ primer, and the genomicDNA of the Enterobacter aerogenes AJ110637 strain as the template toobtain a ybjL gene fragment (B) (SEQ ID NO: 86).

PCR was performed by using the fragments (A) and (B) as templates, andthe primers of SEQ ID NOS: 81 and 85 to obtain a gene fragment (C) withthe fragments (A) and (B) ligated to each other. This gene fragment (C)was blunt-ended by using TaKaRa BKL Kit (Takara Bio), and the 5′ end wasphosphorylated. Then, the fragment was digested with the restrictionenzyme SmaI, and the product was ligated with the plasmid vector pMW218dephosphorylated with alkaline phosphatase to construct a plasmidpMW218::Pthr::Ent-ybjL for ybjL amplification.

The aforementioned vector pMW218::Pthr::Ent-ybjL for amplification ofthe ybjL gene derived from the Enterobacter aerogenes AJ110637 strain,and the control plasmid pMW218 were each introduced into theEnterobacter aerogenes AJ110637ΔadhE/pSTV28::Pthr::pyc strain byelectroporation to obtain transformants which are resistant totetracycline, chloramphenicol and kanamycin. The ybjL-amplified strainderived from the Enterobacter aerogenes AJ110637ΔadhE/pSTV28::Pthr::pycstrain was designated asAJ110637ΔadhE/pSTV28::Pthr::pyc/pMW218::Pthr::Ent-ybjL, and the controlstrain as with pMW218 was designated asAJ110637ΔadhE/pSTV28::Pthr::pyc/pMW218.

<9-4> Effect of Amplification of ybjL Derived from Enterobacteraerogenes in Enterobacter Bacterium

AJ110637ΔadhE/pSTV28::Pthr::pyc/pMW218::Pthr::Ent-ybjL, andAJ110637ΔadhE/pSTV28::Pthr::pyc/pMW218 were each uniformly applied to anLB plate containing 50 μg/ml of kanamycin, 25 μg/ml of tetracycline, and40 μg/ml of chloramphenicol, and cultured at 31.5° C. for 16 hours.Then, the cells were inoculated into 3 ml of a seed medium (20 g/l ofBacto tryptone, 10 g/l of yeast extract, 20 g/L of NaCl) in a test tube,and cultured at 31.5° C. for 16 hours with shaking. A succinic acidproduction medium (100 g/l of glucose, 50 g/L of calcium carbonatesubjected to hot air sterilization for 3 hours or more) in a volume of 3ml was added to the medium obtained above, then the tube was sealed witha silicone stopper, and culture was performed at 31.5° C. for 24 hourswith shaking. After the culture, the amount of succinic acid which hadaccumulated in the medium was analyzed by liquid chromatography, TwoShinn-pack SCR-102H (Shimadzu) connected in series were used as thecolumn, and a sample was eluted at 50° C. with 5 mM p-toluenesulfonicacid. The eluate was neutralized with 20 mM Bis-Tris aqueous solutioncontaining 5 mM p-toluenesulfonic acid and 100 μM EDTA, and succinicacid was quantified by measuring electric conductivity with CDD-10AD(Shimadzu).

The amounts of succinic acid which had accumulated after 24 hours areshown in Table 8.

Succinic acid accumulation and succinic acid yield based on consumedglucose were markedly increased in the ybjL gene-amplified strain

110637ΔadhE/pSTV28::Pthr::pyc/pMW218::Pthr::Ent-ybjL as compared tocontrol AJ110637ΔadhE/pSTV28::Pthr::pyc/pMW218.

TABLE 8 AJ110637ΔadhE/ AJ110637ΔadhE/ pSTV28::Pthr::pyc/pSTV28::Pthr::pyc/ pMW218 pMW218::Pthr::Ent-ybjL Consumed glucose  9.53(±1.85)  9.10 (±0.66) amount (g/L) OD (600 nm)  8.90 (±0.18)  8.72(±0.95) Succinic acid  3.40 (±0.56)  6.37 (±0.51) accumulation (g/L)Succinic acid yield 35.80 (±1.82) 69.70 (±1.79) based on consumedglucose (%)

Explanation of Sequence Listing

SEQ ID NO: 1: ybjL gene of Pantoea ananatis

SEQ ID NO: 2: YbjL of Pantoea ananatis

SEQ ID NO: 3: ybjL gene of Escherichia coli

SEQ ID NO: 4: YbjL of Escherichia coli

SEQ ID NO: 5: Consensus sequence of YbjL of Pantoea ananatis andEscherichia coli

SEQ ID NO: 6: Primer for ams gene disruption

SEQ ID NO: 7: Primer for ams gene disruption

SEQ ID NO: 8: Primer for amplification of ybjL of P. ananatis

SEQ ID NO: 9: Primer for amplification of ybjL of P. ananatis

SEQ ID NO: 10: Sequence of RSFCPG plasmid

SEQ ID NO: 11: Primer for amplification of ybjL of E. coli W3110

SEQ ID NO: 12: Primer for amplification of ybjL of E. coli W3110

SEQ ID NO: 13: Primer for deletion of ldhA

SEQ ID NO: 14: Primer for deletion of ldhA

SEQ ID NO: 15: Primer for confirming deletion of ldhA

SEQ ID NO: 16: Primer for confirming deletion of ldhA

SEQ ID NO: 17: Primer for deletion of lldD

SEQ ID NO: 18: Primer for deletion of lldD

SEQ ID NO: 19: Primer for confirming deletion of lldD

SEQ ID NO: 20: Primer for confirming deletion of lldD

SEQ ID NO: 21: Threonine promoter sequence

SEQ ID NO: 22: Primer for amplification of ybjL of E. coli MG1655

SEQ ID NO: 23: Primer for amplification of ybjL of E. coli MG1655

SEQ ID NO: 24: ybjL gene of Salmonella typhimurium

SEQ ID NO: 25: YbjL of Salmonella typhimurium

SEQ lD NO: 26: ybjL gene of Yersinia pestis

SEQ ID NO: 27: YbjL of Yersinia pestis

SEQ ID NO: 28: ybjL gene of Erwinia carotovora

SEQ ID NO: 29: YbjL of Erwinia carotovora

SEQ ID NO: 30: ybjL gene of Vibrio cholerae

SEQ ID NO: 31: YbjL of Vibrio cholerae

SEQ ID NO: 32: ybjL gene of Aeromonas hydrophia

SEQ ID NO: 33: YbjL of Aeromonas hydrophia

SEQ ID NO: 34: ybjL gene of Photobacterium profundum

SEQ ID NO: 35: YbjL of Photobacterium profundum

SEQ ID NO: 36: ldhA gene of Escherichia coli

SEQ ID NO: 37: LdhA of Escherichia coli

SEQ ID NO: 38: lldD gene of Escherichia coli

SEQ ID NO: 39: LldD of Escherichia coli

SEQ ID NO: 40: Nucleotide sequence of hisD gene of Pantoea ananatis

SEQ ID NO: 41: Primer for amplification of fragment for incorporation ofKm^(r) gene into hisD gene

SEQ ID NO: 42: Primer for amplification of fragment for incorporation ofKm^(r) gene into hisD gene

SEQ ID NO: 43: Primer for amplification of cat gene

SEQ ID NO: 44: Primer for amplification of cat gene

SEQ ID NO: 45: Primer for amplification of sacB gene

SEQ ID NO: 46: Primer for amplification of sacB gene

SEQ lD NO: 47: Primer for amplification of DNA fragment containingPlacUV5 promoter

SEQ lD NO: 48: Primer for amplification of DNA fragment containingPlacUV5 promoter

SEQ ID NO: 49: Primer for amplification of DNA fragment containingλRedαβγ gene and tL3

SEQ ID NO: 50: Primer for amplification of DNA fragment containingλRedαβγ gene and tL3

SEQ ID NO: 51: Primer for amplification of DNA fragment containingPlacUV5 promoter and TrrnB

SEQ ID NO: 52: Primer for amplification of DNA fragment containingPlacUV5 promoter and TrrnB

SEQ ID NO: 53: Primer for amplification of attL

SEQ ID NO: 54: Primer for amplification of attL

SEQ ID NO: 55: Nucleotide sequence of attL

SEQ ID NO: 56: Primer for amplification of attR

SEQ ID NO: 57: Primer for amplification of attR

SEQ ID NO: 58: Nucleotide sequence of attR

SEQ ID NO: 59: Primer for amplification of DNA fragment containing blagene

SEQ ID NO: 60: Primer for amplification of DNA fragment containing blagene

SEQ ID NO: 61: Primer for amplification of DNA fragment containingter_rrnB

SEQ ID NO: 62: Primer for amplification of DNA fragment containingter_rrnB

SEQ lD NO: 63: Nucleotide sequence of the DNA fragment containingter_thrL terminator

SEQ ID NO: 64: Primer for amplification of DNA fragment containingter_thrL terminator

SEQ ID NO: 65: Primer for amplification of DNA fragment containing ter_thrL terminator

SEQ ID NO: 66: ams operon of Pantoea ananatis

SEQ ID NO: 67: AmsH of Pantoea ananatis

SEQ ID NO: 68: AmsI of Pantoea ananatis

SEQ ID NO: 69: AmsA of Pantoea ananatis

SEQ ID NO: 70: AmsC of Pantoea ananatis

SEQ ID NO: 71: AmsB of Pantoea ananatis

SEQ ID NO: 72: Nucleotide sequence of primer for deletion of adhE

SEQ ID NO: 73: Nucleotide sequence of primer for deletion of adhE

SEQ ID NO: 74: Nucleotide sequence of adhE of Enterobacter aerogenesAJ110637 partial sequence)

SEQ ID NO: 75: Primer for amplification of threonine promoter

SEQ ID NO: 76: Primer for amplification of threonine promoter

SEQ ID NO: 77: Threonine promoter gene fragment

SEQ ID NO: 78: Primer for amplification of pyruvate carboxylase gene

SEQ ID NO: 79: Primer for amplification of pyruvate carboxylase gene

SEQ ID NO: 80: Pyruvate carboxylase gene fragment

SEQ ID NO: 81: Primer for amplification of threonine promoter

SEQ ID NO: 82: Primer for amplification of threonine promoter

SEQ ID NO: 83: Threonine promoter gene fragment

SEQ ID NO: 84: Nucleotide sequence of primer for amplification of ybjLof Enterobacter aerogenes AJ110637

SEQ ID NO: 85: Nucleotide sequence of primer for amplification of ybjLof Enterobacter aerogenes AJ110637

SEQ ID NO: 86: Nucleotide sequence of ybjL of Enterobacter aerogenesAJ110637

SEQ ID NO: 87: Amino acid sequence of YbjL of Enterobacter aerogenes AJ110637

SEQ ID NO: 88: Consensus sequence of YbjL of Escherichia, Pantoea andEnterobacter

INDUSTRIAL APPLICABILITY

Production efficiency of an acidic substance having a carboxyl group canbe improved by using the microorganism of the present invention.

While the invention has been described in detail with reference topreferred embodiments thereof, it will be apparent to one skilled in theart that various changes can be made, and equivalents employed, withoutdeparting from the scope of the invention. Each of the aforementioneddocuments is incorporated by reference herein in its entirety.

1. A method for producing an acidic substance having a carboxyl group comprising culturing in a medium a microorganism having an ability to produce an acidic substance having a carboxyl group to produce and accumulate the acidic substance having a carboxyl group in the medium, and collecting the acidic substance having a carboxyl group from the medium, wherein the acidic substance is L-glutamic acid and/or L-aspartic acid, wherein the microorganism has been modified to enhance expression of an ybjL gene, and wherein the ybjL gene encodes a protein selected from the group consisting of: (A) a protein comprising the amino acid sequence of SEQ ID NO: 2; and (B) a protein comprising a variant of the amino acid sequence of SEQ ID NO: 2, wherein one to 5 amino acid residues are substituted, deleted, inserted or added, and wherein, upon expression, said variant improves the ability of the microorganism to produce an acidic substance having a carboxyl group.
 2. The method according to claim 1, wherein said enhanced expression is obtained by a method selected from the group consisting of: A) increasing copy number of the ybjL gene, B) modifying an expression control sequence of the ybjL gene, and C) combinations thereof.
 3. The method according to claim 1, wherein the microorganism is a bacterium belonging to the family Enterobacteriaceae.
 4. The method according to claim 3, wherein the bacterium belongs to a genus selected from the group consisting of Escherichia, Enterobacter, Raoultella, Pantoea, and Klebsiella.
 5. The method according to claim 1, wherein the microorganism is a rumen bacterium.
 6. The method according to claim 5, wherein the microorganism is Mannheimia succiniciproducens. 